Download ABI™ 3948 Reference Manual

ABI 3948
™
Nucleic Acid Synthesis and Purification System
Reference Manual
© Copyright 2001, 2011 Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
ABI PRISM and the ABI PRISM design, Applied Biosystems, FastPhoramidite, ONESTEP, OPC, Phosphalink, and POLYPORE are registered trademarks
of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
ABI, CATALYST, LV40, and PDQ are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
All other trademarks are the sole property of their respective owners.
Contents
1 Introduction to the Reference Manual. . . . . . . . . . . . . . . . . . . . . 1-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
Using This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
User Bulletins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Key Terms Defined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
2 System Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Section 1 - General Description of Processes, System Control, and Hardware . . . . . . . . . 2-2
Basic Instrument Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
General Description of Instrument Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
System Control and Sensing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Hardware Description Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
Section 2 - More Details of System Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8
OneStep Column and Column Turntable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9
Jaw Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11
Pressure Regulation and Control (PRC) Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
Synthesis Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13
Cleavage Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15
Deprotection Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16
Purification Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
Quantitation Module. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-20
Sample Collection Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21
3 Automated DNA Synthesis Chemistry . . . . . . . . . . . . . . . . . . . . . 3-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Section 1 – DNA Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
DNA Synthesis Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
Overview of the Phosphoramidite Method of Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
Detritylation Chemistry Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Coupling Chemistry Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11
Capping Chemistry Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13
Oxidation Chemistry Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15
DNA Cleavage and Deprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18
iii
Section 2 – Oligonucleotide Purification, Quantitation, and Storage . . . . . . . . . . . . . . .
DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview of Oligonucleotide Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Measurement of ODU and Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storage of the Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alternative Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3-19
3-20
3-22
3-23
3-27
3-28
4 Overview of Software Commands . . . . . . . . . . . . . . . . . . . . . . . . 4-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Section 1 – File and Edit Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Overview of File Menu Commands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3
New Synthesis Order Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
Open Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8
Open Synthesizer Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10
Other File Menu Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Overview of Edit Menu Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-17
Section 2 – Synthesizer and Window Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-20
Overview of Synthesizer Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-21
Abort Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Interrupt Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Resume Command. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pause After Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Synchronize Clocks Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Change Password Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Change Name Command. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument Preferences Command. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Window Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Section 3 – RunFiles, Sample Labeling, Synthesis Order and Multi-Order Files . . . . .
RunFiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Labeling Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Label View Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About Synthesis Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multi-Order Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4-22
4-22
4-22
4-23
4-24
4-25
4-26
4-27
4-30
4-31
4-32
4-33
4-36
4-38
4-41
5 Communication View and Operational Views . . . . . . . . . . . . . . . 5-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Section 1 - General Information about Operational Synthesizer Views . . . . . . . . . . . . . .
About the Synthesizer Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Communication View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run Setup View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run Protocol View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv
5-1
5-2
5-4
5-6
5-7
5-9
Monitor Chemistry View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11
Monitor Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15
Liquid Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16
Monitor Run View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-19
Section 2 - Using the Run Setup View and Extending a Run . . . . . . . . . . . . . . . . . . . . . . 5-20
Importing Synthesis Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-21
Selecting Orders for the Next Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-23
Synthesis Order Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-24
Assigning Chemistry and
AutoSorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-25
Bottle Usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-27
Using the Load Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-28
Extending a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-30
6 Synthesizer Views Supporting Operation . . . . . . . . . . . . . . . . . . . 6-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1
Section 1 – Non-Cycle Operational Support Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
Manual Control View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3
Power Fail View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6
Instrument Test View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7
B+ Tet Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8
Reagent Utilization Table View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9
Section 2 – Editing Cycle and Procedure Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11
Edit Cycle and Procedure Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-12
Edit Synthesis Cycle View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17
Edit Cleavage Cycle View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-18
Edit Purification Cycle View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19
Edit Begin Procedure View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20
Edit End Procedure View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21
Edit Bottle Procedure View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22
Misc (Miscellaneous) Procedures View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23
A Valves and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Information on ABI 3948 System Chemistry Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3
Valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5
Overview of Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7
Valve Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10
Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11
Valve Functions by Functional Category . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-15
Non-Valve Hardware Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-23
v
B ABI 3948 System Special Functions . . . . . . . . . . . . . . . . . . . . . . B-1
In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
Overview of Special Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2
Listing of Special Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5
C Cycles, Procedures, and System Messages . . . . . . . . . . . . . . . . . C-1
In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1
3948 Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2
The Standard Synthesis Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3
The Standard Cleavage/Deprotection Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-9
The Standard Purification Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-18
Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-32
System Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-38
D 3948 System Function List and Other Cycles . . . . . . . . . . . . . . . D-1
In This Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1
ABI 3948 System Function List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2
The Purification Dye Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-39
The Purification Biotin Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-48
E Instrument Plumbing Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
F Limited Warranty Statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1
Index
vi
Introduction to the
Reference Manual
1
1
In This Chapter
User and Reference The ABI 3948 System Reference Manual is one of two manuals in the document set
Manuals supporting the ABI™ 3948 Nucleic Acid Synthesis and Purification System. The first
manual in the set, the ABI 3948 System User’s Manual, is intended for daily operation,
while this reference manual presents detailed information needed to fully understand,
use, and maintain the instrument.
Topics Covered This chapter contains the following topics:
Topic
Using This Manual
Contents of the Manual
User Bulletins
Purpose of User Bulletins
Key Terms Defined
Table of Key Terms
See page
1-2
1-2
1-4
1-4
1-4
1-4
Introduction to the Reference Manual 1-1
Using This Manual
Contents of the This manual contains five chapters and several appendices.
Manual
Chapter/
Appendix
1
2
3
4
5
1-2 Introduction to the Reference Manual
Title
Introduction to the
Reference Manual/
Instrument
System Description
Chemistry for
Automated DNA
Chemistry
Overview of System
Commands
Communication View
and Operational
Views
Types of Information
♦
Purposes of User and Reference manuals
♦
Information about safety and user bulletins
♦
Description of the ABI 3948 System
♦
Overview of how the system is controlled
♦
Descriptions of the six modules:
–
Synthesis
–
Cleavage
–
Deprotection
–
Purification
–
Quantitation
–
Sample Collection
♦
DNA synthesis overview
♦
Description of Cleavage and Deprotection
♦
Quantitation of the Oligonucleotide
♦
Purification
♦
Storage of the Oligonucleotide
Detailed command information covering the following
menus:
♦
File menu
♦
Open Synthesizer menu
♦
Edit menu
♦
Synthesizer menu
♦
Window menu
Detailed information covering the following:
♦
Synthesizer Window
♦
Communication view
♦
Run Setup view
♦
Run Protocol view
♦
Monitor Chemistry view
♦
Monitor Instrument view
♦
Monitor Run view
Chapter/
Appendix
6
A
B
C
Title
Other Synthesizer
Views
Valves and Functions
Cycles, Procedures,
and System
Messages
3948 System Function
List and Other Cycles
Types of Information
Detailed information covering the following:
♦
MISC (Miscellaneous) Procedures view
♦
Manual Control view
♦
Power Fail view
♦
Instrument Test view
♦
B+Tet Calibration view
♦
Reagent Utilization Table view
♦
Editing Cycle and Procedure views
♦
Default Contents of the B+ Tet Calibration View
♦
Default Contents of the Reagent Utilization View
Detailed information on the following:
♦
Standard Synthesis Cycle
♦
Standard Cleavage/Deprotection Cycle
♦
Standard Purification Cycle
♦
Procedures
♦
System Messages
♦
3948 System Function List
♦
Purification Dye Cycle
♦
Purification Biotin Cycle
Introduction to the Reference Manual 1-3
User Bulletins
Purpose of User User Bulletins (UBs) contain technical information that is essential to ABI 3948
Bulletins instrument operation and related laboratory techniques. UBs are the quickest way to
ensure that you have current information. They are produced periodically and mailed
to you as they become available.
Please read the UBs before operating your ABI 3948 instrument. Current UBs are
found under their own tab at the end of the ABI 3948 Instrument Reference Manual.
Key Terms Defined
Table of Key Terms The following table lists the key terms needed for operation of the 3948.:
Term
Definition
3948Control Views
Protocol
Contains the three chemistry cycles needed to produce an
oligonucleotide:
♦
Synthesis cycle
♦
Cleavage cycle
♦
Purification cycle
When new chemistry cycles are available, you can assign them to a new
protocol using the Run Protocol view. For more information on creating
a new protocol with your own cycles, see Chapter 6 of this manual.
Begin and End
Procedures
These procedures, chosen during Run Setup, are run before and after
oligonucleotide production. If you create a new protocol with your own
cycles, you may need to create revised versions of these procedures
(see Chapter 6 for information on revising these procedures).
Commands
Abort
Immediately terminates execution of a run or Manual Control action in
progress.
Interrupt
Halts the instrument at the first safe step for all active chemistries
(synthesis, cleavage, purification, and procedures).
There are two ways to initiate an interrupt:
♦
Choose Interrupt from the Synthesizer menu
♦
Press the Interrupt button on the ABI 3948 Synthesizer Front Panel
If you use Interrupt to halt the instrument operation, use the Resume
command from the Synthesizer menu to restart the instrument
Pause After
Halts the instrument after the designated synthesis, allowing the user to
extend the run or do a bottle change if necessary.
To initiate a Pause After, choose Pause After from the Synthesizer
menu.
1-4 Introduction to the Reference Manual
System Description
2
2
In This Chapter
Topics Covered This chapter provides a general description of instrument chemistry processes and
hardware.
The chapter contains two sections:
Topic
See page
General Description of Processes, System Control, and Hardware
2-2
More Details of System Hardware
2-8
System Description 2-1
Section 1 - General Description of Processes, System Control, and Hardware
Topics Covered This section provides a general description of instrument Chemistry and other
processes, system control and sensing, and an overview of instrument hardware.
The section covers the following topics:
Basic Instrument Features
2-3
Phosphoramidite Method of Synthesis
2-3
Pressure-Driven Chemical Delivery
2-3
Macintosh 3948Control Software
2-3
General Description of Instrument Chemistry
2-4
Automation of Chemistry Processes
2-4
The First Four Chemistry Processes
2-4
Quantitation
2-5
Sample Collection
2-5
System Control and Sensing
2-6
Introduction
2-6
Types of Storage
2-6
Electromechanical and Electrical Drivers
2-6
Gas Pressure Sensors/Sensor Drivers
2-6
Liquid Sensors
2-6
Hardware Description Overview
Types of Hardware Described
2-2 System Description
2-3
Automatic Oligonucleotide Production
2-7
2-7
Basic Instrument Features
Automatic The ABI 3948 Nucleic Acid Synthesis and Purification system completely automates
Oligonucleotide the entire process of oligonucleotide production: synthesis, cleavage, deprotection,
Production purification, quantitation, and sample collection. When used as a system utilizing ABI
reagents and columns, this instrument produces high quality synthetic DNA while
minimizing synthesis time and cost.
Phosphoramidite The phosphoramidite method of oligonucleotide synthesis is used because of its
Method of Synthesis inherently high coupling efficiency and the stability of the starting materials. The 3´
terminal nucleoside attached to a solid support, which is contained within a disposable
column (the OneStep™ column). Nucleoside bases are added one at a time to the
support-bound DNA chain until the sequence is fully synthesized. Solid support
synthesis allows excess reagents to be removed by filtration and eliminates the need
for purification between base additions.
Pressure-Driven Applied Biosystems synthesizers use a pressure driven chemical delivery system to
Chemical Delivery deliver reagents and solvents to a reaction column chamber (OneStep column).
Reagent and solvent deliveries also rely on our zero-dead volume valves which
increase reliability, eliminate cross-contamination and reduce cycle costs.
ABI 3948 System You can program cycles, functions, and procedures for use in the synthesizer from the
Software for ABI 3948 system software for Macintosh computer. Once you download chemistry
Macintosh protocols, an internal controller/driver within the synthesizer exercises real-time
control of the instrument. The ABI 3948 instrument can run preprogrammed protocols
or you can create customized cycles. The Macintosh software is also used to fill out
the Synthesis Orders used as sequence input for the instrument.
System Description 2-3
General Description of Instrument Chemistry
Automation of The ABI™ 3948 DNA Synthesis and Purification system automates four processes of
Chemistry Processes DNA chemistry (synthesis, cleavage, deprotection, and purification), and then
quantitates and automatically collects the DNA product.
The synthesis, cleavage, and purification processes are implemented in three
modules that operate concurrently to provide the high throughput capacity of the
instrument—as many as 48 twenty-mer oligonucleotides per day of operation.
The First Four Three of the four chemistry processes (synthesis, cleavage, and purification) take
Chemistry Processes place within the OneStep™ column as it moves between the three modules. The fourth
process, deprotection, takes place outside the OneStep column in the deprotection
coils between cleavage and purification.
Module
Process
Synthesis module
The first process occurs in the column while it is positioned at
the Synthesis module. The OneStep column contains the
3´-terminal nucleoside, covalently attached to support
material. The DNA chain is built by adding one base at a time
to the support-bound nucleoside.
IMPORTANT
You can synthesize crude oligonucleotides
with the 5´ trityl group either on or off; you must synthesize
purified oligonucleotides with the 5´ trityl group on.
Cleavage module
During the second process of automated oligonucleotide
production, the OneStep column is positioned at the
Cleavage module and processed as follows:
a.
In the Cleavage module a delivery of concentrated
aqueous ammonium hydroxide cleaves the
oligonucleotide from the support.
b.
The DNA product in the ammonium hydroxide solution is
then transferred to the Deprotection module for
processing outside the column.
Deprotection module
In the Deprotection module, protecting groups are removed
from the oligonucleotide as the third process by heating the
solution containing the oligonucleotide for one hour at 65 °C.
Purification module
During the fourth DNA chemistry process, purification, the
desired, full-length oligonucleotide is separated from failure
sequences and other impurities at the Purification module.
Purification is performed as follows:
I
2-4 System Description
a.
The deprotected product is passed in ammonium
hydroxide through the support in the original OneStep
column.
b.
The tritylated, full-length oligonucleotide binds to the
support in the column while detritylated failure
sequences and other impurities are washed away.
c.
Following detritylation, the DNA products are eluted
from the OneStep column, using a solution of 20%
acetonitrile in water
Quantitation In the fifth production process, the purified oligonucleotides are quantitated using a
UV (ultraviolet) light detection system as follows.
♦
The product, in a 20% acetonitrile solution, passes through the detector as it is
delivered to the sample collection module.
♦
The absorbance is reported in ODUs (Optical Density Units) and picomoles to
indicate quantity.
Sample Collection Automated oligonucleotide production concludes with the collection of the DNA
sample in the sample collection module:
♦
The sample collection tray contains 48 sample vials to allow unattended collection
of 48 DNA samples.
♦
A septum sheet under the OligoRack™ cover minimizes evaporation from and
contamination of the sample vials.
♦
Each of the sample vials, covered by a septum, holds the 1-mL DNA sample
volume from a OneStep column obtained in either the deprotection or purification
module. The vial septum is chemically inert to both 20% acetonitrile/H2O and
ammonium hydroxide.
System Description 2-5
System Control and Sensing
Introduction The ABI™ 3948 DNA Synthesis and Purification System is controlled from a computer
with the Macintosh-compatible ABI 3948 instrument operating software. A user
programs the system through the computer which interacts with embedded software
on the instrument.
Types of Storage Several types of memory storage are available on the instrument:
♦
The embedded software code allows real-time control via the Macintosh®
computer.
♦
The 3948 boot ROM holds instrument-unique data such as the instrument serial
number.
♦
Battery backed-up memory stores critical data, such as power-failure recovery
information and any data that is not transferred to the Macintosh.
♦
Temporary memory is reserved for the proper and reliable operation of the
instrument. It is possible to increase synthesizer memory to accommodate future
upgrades.
Electromechanical The instrument contains a number of electromechanical drivers that control the
and Electrical process of oligonucleotide production:
Drivers ♦ One of these, the driver for the OneStep column turntable, properly positions the
OneStep columns in the jaw mechanisms.
♦
Another driver, for the jaw mechanism, engages and disengages the jaw module
to ensure a leak-tight seal.
♦
Other driver mechanisms include: the deprotection heater driver, the sample
collection driver, and valve drivers.
Gas Pressure Gas pressure sensors ensure accurate pressure-regulated delivery rates. In addition
Sensors/Sensor to regulation of gas and liquid flows, sensor drivers monitor the following instrument
Drivers operations:
♦
Maintenance of the battery supported memory
♦
Positioning and movement of the OneStep column turntable and the jaw
mechanism
♦
Regulation of the deprotection system temperature
♦
Sample collection
♦
Regulation and usage of the pressure management system that provides
automated leak test capability
♦
Valve driver power usage and load/no-load testing to detect solenoid shorts and
disconnections
Liquid Sensors The instrument also has liquid sensors to ensure that adequate amounts of reagents
are available and properly delivered during the run. Liquid sensors are shown in the
figure on page 2-16.
2-6 System Description
Hardware Description Overview
Types of Hardware The following hardware is described in this chapter:
Described ♦ OneStep™ Column and Column Turntable
Four types of OneStep columns are used. One for each of the starting monomers.
The turntable is used to hold the columns and move them between the three
chemistry jaw mechanisms (Synthesis, Cleavage, and Purification).
♦
Jaw Mechanism
This is the device used to clamp on the top and bottom of OneStep columns and
provide flow paths for chemistry.
♦
Pressure Regulation and Control Module (PRC)
This module regulates and controls various pressures in the system.
♦
Synthesis Module
This module consists of the Synthesis jaw mechanism and associated plumbing.
♦
Cleavage Module
This module consists of the Cleavage jaw mechanism and associated plumbing.
♦
Deprotection Module
This module consists of deprotection coils and associated plumbing.
♦
Purification Module
This module consists of the Purification jaw mechanism and associated plumbing.
♦
Quantitation Module
This module consists of the UV detector and associated plumbing.
♦
Sample Collection Module
This module consists of the following three components:
–
A mechanism and tray to hold the OligoRack/red rack and waste bottles.
The mechanism moves the tray to position tubes under the needle delivery
system and extend the tray so that individual tubes can be removed.
–
An OligoRack or optional red rack to hold 1.2-mL tubes.
–
A needle delivery system to individually delivery synthesized or full length
oligonucleotides to assigned positions.
System Description 2-7
Section 2 - More Details of System Hardware
This section provides general information on system hardware.
OneStep Column and Column Turntable
OneStep Columns and Column Turntable
OneStep Column Turntable
Jaw Mechanism
2-9
2-10
2-11
Uses of the Three Jaw Mechanisms
2-11
Pressure Regulation and Control (PRC)
2-12
Characteristics of Module
Synthesis Module
2-12
2-13
Location on the Plumbing Diagram
2-13
The Process of Synthesis
2-14
Cleavage Module
2-15
Location on the Plumbing Diagram
2-15
The Process of Cleavage
2-15
Deprotection Module
2-16
Deprotection Module Components
2-16
Location on the Plumbing Diagram
2-16
The Process of Deprotection
2-17
Purification Module
2-18
Location on the Plumbing Diagram
2-18
The Process of Purification
2-19
Quantitation Module
Description of UV Detection System
Sample Collection Module
2-8 System Description
2-9
2-20
2-20
2-21
Three Main Components
2-21
Two Types of Racks
2-22
Waste Containers
2-22
OneStep Column and Column Turntable
OneStep Column The OneStep column, shown in orthogonal and cross-sectional views below, contains
a proprietary support composed of a highly cross-linked polystyrene with the pore and
particle sizes appropriate for both synthesis and purification.
Orthogonal view
Cross-sectional view
GR0211•Model 3948 Refernce Manual
Orthographic view of Column
Synthesis
= Synthesis Powder
support
Filter
(frit) material
= Filter Material
(pore size)
The OneStep column has these characteristics:
♦
Color coded to identify the starting monomer (A=green, G=yellow, C=red, and
T=blue)
♦
Accommodates 40 nmol of pure DNA product
♦
Easy to load and remove from the column tray
♦
Chemically inert to all chemicals used on the ABI 3948 and is designed to provide
a leak-free seal with the synthesis, cleavage, and purification modules
System Description 2-9
OneStep Column The OneStep column turntable, shown below, does the following:
Turntable ♦ Positions the columns in the synthesis, cleavage, and purification
modules to ensure that the jaw mechanisms at these modules form a
leak-free seal.
♦
Holds 48 columns and is easy to load.
♦
Precisely indexes the position of each column on the turntable for
software control of each step of chemical delivery.
Note
For clarity, the turntable in the figure is shown pulled away from the three jaw
mechanisms with which it is associated. As the turntable rotates, OneStep columns in
all 48 positions are presented in turn to the three jaw mechanisms for reagent
deliveries.
2-10 System Description
Jaw Mechanism
Uses of the Three The jaw mechanism, shown below, is used in conjunction with the OneStep columns
Jaw Mechanisms and turntable to provide a leak-tight seal for the reagent/gas delivery system.
Three jaws, one for each of the three chemistry modules (synthesis, cleavage, and
purification), operate independently so that you can perform all three chemistries
concurrently. All wetted components used in the mechanism are chemically inert to all
reagents used for synthesis, cleavage and purification.
System Description 2-11
Pressure Regulation and Control (PRC) Module
Characteristics of The PRC board assembly function:
Module ♦ Provides accurate gas delivery, regulates pressure to ensure sufficient volumes of
reagents, and closes down delivery at shut off.
♦
Is entirely controlled via firmware and software.
♦
Has 10 individually selectable valves with 3 outlet ports for each valve.
♦
Individually controls the pressure settings of each valve via software. The
pressure can be variably set from 0 psig (off) and up to 12 psig.
Note
♦
2-12 System Description
All used outlet port have a check valve and un-used ports are plugged.
Has the following pressure transducers:
–
Ten low pressure transducers to enable monitoring of pressure in the “Monitor
Instrument” View.
–
One pressure transducer to monitor inlet gas pressure.
Synthesis Module
Location on the The Synthesis module is a station on the column turntable where synthesis is
Plumbing Diagram performed on up to three reaction cartridges at a time. The portion of the plumbing
diagram containing the Synthesis module is shown below. Liquid sensors, like those
indicated in the plumbing diagram, are shown on page 2-16. For the full instrument
plumbing diagram, see Appendix E.
System Description 2-13
The Process of Synthesis is performed by delivering phosphoramidites and reagents through a
Synthesis OneStep column while the column is clamped firmly in a jaw mechanism to ensure
leak free delivery:
♦
Controlled quantities of reagents are supplied step-by-step to the module from
reagent bottles by a system of valve blocks using positive-lift valves and gas/
liquid sensors.
♦
Phosphoramidites are supplied from 2-g bottles and reagents are supplied in the
following bottle sizes:
–
TCA (2L), ACN (4 L)
–
Iodine (450 mL)
–
Acetic Anhydride (450 mL)
–
Tetrazole (450 mL)
–
NMI (450 mL)
Reagent volumes correspond roughly to the rate at which they are consumed during
synthesis.
2-14 System Description
Cleavage Module
Location on the The Cleavage module is a station on the column turntable where cleavage is
Plumbing Diagram performed on up to three columns at a time during the cleavage cycle. The portion of
the plumbing diagram containing the Cleavage module is shown below. Liquid
sensors, like those indicated in the plumbing diagram, are shown in the figure on
page 2-16. For the full instrument plumbing diagram, see Appendix E.
The Process of Cleavage is performed by delivery of concentrated ammonium hydroxide through a
Cleavage OneStep column while the column is clamped firmly in a jaw mechanism to ensure
leak free delivery.
System Description 2-15
Deprotection Module
Deprotection Unlike the Synthesis, Cleavage, and Purification processes, which are performed
Module Components within the OneStep columns, deprotection is performed on the product in solution
outside of these columns. The components of the Deprotection module are shown
below.
Valve solenoids
Deprotection coils
Liquid sensors
Valve blocks
Heater housing block
Location on the The portion of the plumbing diagram containing the Deprotection module is shown
Plumbing Diagram below. For the full instrument plumbing diagram, see Appendix E.
2-16 System Description
The Process of Deprotection includes the following:
Deprotection ♦ Eluting the product in solution from the column in the Cleavage module and
transferring it to one of three coils in the Deprotection module heater (see figure
above).
Note
♦
Liquid sensors monitor the transfer of solution to the coils.
Removing the remaining protecting groups by heating the product in solution for a
programmed period of time at 65 °C.
Note
All wetted components used in the module are chemically inert to ammonium
hydroxide.
System Description 2-17
Purification Module
Location on the Like Synthesis and Cleavage, Purification is performed within the OneStep columns in
Plumbing Diagram this module. Liquid sensors, like those indicated in the plumbing diagram, are shown
in the figure on page 2-16. The portion of the plumbing diagram containing the
Purification module is shown below. For the full instrument plumbing diagram, see
Appendix E.
2-18 System Description
The Process of Purification is performed using a trity-selective purification chemistry to separate the
Purification desired full-length olignonucleotides from failure sequences and impurities:
♦
The deprotected product, dissolved in the cleavage solution (NH4OH), passes
through the columns which contain a purification support.
♦
The tritylated full-length oligonucleotide binds to the support while detritylated
failure sequences and other impurities are washed away.
♦
The 5´-terminal trityl group, which was not removed at the conclusion of synthesis,
is removed with 3% TFA.
♦
The oligonucleotide product is eluted from the column with a solution of 20%
acetonitrile in water.
.
System Description 2-19
Quantitation Module
Description of UV Oligonucleotides are quantitated in a 20% acetonitrile solution using a UV detection
Detection System system:
2-20 System Description
♦
A low-pressure mercury lamp (P/N 100750) in the UV detector, shown below,
provides a UV-light source at 254.5 nm.
♦
As each oligonucleotide passes out of the purification module, through the cuvette
on its way to the sample collection system, current is measured (mV) and then
converted into optical density units and picomoles.
Sample Collection Module
Three Main Samples are collected in the ABI™ 3948 Nucleic Acid Synthesis and Purification
Components System by a sample collector system with three components:
♦
An OligoRack (red rack) or white rack with 48 positions for 1.2-mL tubes
♦
A needle delivery system to individually deliver synthesized or full length
oligonucleotides to assigned positions;
♦
A mechanism that moves the tray so that you can properly remove either an entire
rack or individual tubes from each of the 48 positions (see figure below).
Note
This is a rear three-quarter view of the module. The sample collection tray, which holds
an OligoRack, is shown extended in the figure and by itself below. At the top of the figure is the
sample collector needle assembly.
System Description 2-21
Note
The sample collector tray is used to move an inserted rack into and out of the Sample
Collection module. The two circular openings at the rear are used to hold waste bottles.
Two Types of Racks The two types of racks have the following configurations:
♦
The 4X12 configuration rack that comes with the instrument, the white rack, is not
disposable and cannot be purchased separately (the screw top tubes in the rack
are removed for customers).
♦
The optional red rack may be purchased to enable an entire rack to be shipped to
a single customer. This type of rack has an 8X6 configuration with included vials
and press tops.
The two racks measure 8.5 x 12.7 x 5.5 cm and are shown in the Sample Collector
tray below:
Position #48
10-mL sample collector
waste bottles positions
key
Position #48
Position #1
Position #1
Optional Red Rack with 6 x 8
format - uses press top vials
Note
Rack with 4 x12 format
(white - uses screw top tubes)
The red rack contains 6 rows of 8 tubes alternating with 6 rows of tube caps.
Each of the two types of racks contains 48 sample positions and has the following
characteristics:
♦
One corner of each rack cover is beveled. When positioned correctly on the
sample collection tray, the beveled corner faces the inside of the instrument.
♦
Each rack cover is lined with a septum to minimize evaporation and
contamination.
♦
The tubes and all wetted parts of the system are chemically inert to the 20%
acetonitrile solution used to elute oligonucleotides and to concentrated
ammonium hydroxide.
Waste Containers Two empty bottles serve as waste collection vials as follows:
♦
Between samples, the collection needle is rinsed.
♦
The rinses are delivered to one of the two waste bottles.
♦
You must empty these waste bottles after each 3948 run. Bottles are located in
the circular opening shown in the figure above.
IMPORTANT
Refer to the Chemical Safety section of the ABI™ 3938 DNA Synthesizer Site
Preparation and Safety Guide for a description of the contents of this collected waste.
2-22 System Description
Automated DNA
Synthesis Chemistry
3
3
In This Chapter
Topics Covered This chapter presents a description of DNA synthesis chemistry. In addition, it
contains an overview of oligonucleotide analysis and purification procedures, and
information about quantifying and storing oligonucleotides.
This chapter contains two sections:
Section
See page
DNA Chemistry
3-2
Oligonucleotide Quantitation, Purification, and Storage
3-19
Automated DNA Synthesis Chemistry 3-1
Section 1 – DNA Chemistry
Topics Covered This section describes how DNA synthesis chemistry is implemented on the solid
support within the OneStep™ column on the ABI™ 3948 Nucleic Acid Synthesis and
Purification System.
The section covers the following topics:
Topic
DNA Synthesis Overview
See page
3-3
DNA Synthesis Process
3-3
DNA Synthesis Cycle
3-3
Solid Support
3-3
Completion of the Synthesis Cycle
3-5
Overview of the Phosphoramidite Method of Synthesis
3-6
Four Chemistry Reactions
3-6
Chemistry of Choice
3-7
Phosphoramidite Nucleotides
3-7
Functional Groups
3-8
Detritylation Chemistry Reactions
Detritylation Process
Depurination
Coupling Chemistry Reactions
Washing and Drying of Support
Simultaneous Delivery of Phosphoramidites and Tetrazole
Capping Chemistry Reactions
3-9
3-9
3-9
3-11
3-11
3-11
3-13
Why Capping is Needed
3-13
Capping Process
3-13
Oxidation Chemistry Reactions
Oxidation Reaction
Importance of Oxidation After Capping
DNA Cleavage and Deprotection
3-15
3-15
3-15
3-16
Introduction
3-16
Cleavage
3-16
Phosphate Deprotection
3-16
Base Deprotection
3-17
References
3-18
3-2 Automated DNA Synthesis Chemistry
DNA Synthesis Overview
DNA Synthesis In DNA synthesis, synthesis proceeds as follows:
Process
Stage
Description
1
A reactive 3´ phosphorus group of one nucleotide (in the form of a monomer in
solution) is coupled to the 5´ hydroxyl of a nucleotide immobilized on a solid
support.
2
An internucleotide linkage forms as a result of the coupling.
3
Three more chemical reactions follow to prepare the growing chain of DNA for the
next coupling.
In each DNA synthesis cycle, one nucleotide monomer is added to the chain.
DNA Synthesis Cycle Each cycle of nucleotide addition consists of four steps, which differ depending on the
desired structure of the final product.
♦
Detritylation
♦
Coupling
♦
Capping
♦
Oxidation
These reaction steps are repeated in the same order until all nucleotides in the
sequences have been added. The operator of the DNA synthesizer defines the
desired sequence and length of the final product.1 Following synthesis, the
oligonucleotide is cleaved and deprotected from the solid support.
Solid Support The ABI™ 3948 Nucleic Acid Synthesis and Purification System uses a solid-phase
synthesis chemistry with a highly cross-linked polystyrene support and attaches the
nucleotide 3´-hydroxyl to the support with an aminomethyl linker.
The following support characteristics are described in this subsection:
♦
Solid Phase Synthesis Chemistry
♦
Highly Cross-linked Polystyrene Support
♦
Attachment of nucleotide 3´-hydroxyl to Support by Linker
Solid Phase Synthesis Chemistry
The ABI 3948 DNA Synthesizer uses solid phase chemistry:
Stage
Description
1
Before beginning a synthesis, one of the support-bound nucleotides (A, C, G, or T)
contained within a OneStep™ column, is placed on the instrument.
2
As synthesis proceeds:
♦
All reagents and solvents flow through the support, which is contained within a
unique synthesis/ purification column
♦
The growing DNA chain remains covalently attached to the insoluble synthesis
support within the column.
Automated DNA Synthesis Chemistry 3-3
Highly Cross-linked Polystyrene Support
The support used for DNA synthesis/purification is a highly cross-linked polystyrene
with the following characteristics during major phases of chemistry:
During…
The support…
Synthesis
produces coupling efficiencies of about 98% and higher, as measured by the
trityl cation assay.
Purification
is also used as a purification media for the DNA.
Note
The support can be used for purification because the polystyrene
used is a porous, non-swelling bead which exhibits hydrophobic properties.
Attachment of Nucleotide 3´-hydroxyl to Support by Linker
As shown in the figure below, polystyrene has an aminomethyl linker attached to its
surface. This type of linker enables the support characteristics listed in the table which
follows the figure.
P
B
DMTO
O
B
P
O C C H2CH 2CNHCH2
O
=A
bz
G dmf
bz
C
T
polystyrene
O
Note
The DMT-protected nucleotide is attached to the polystyrene support (B = Base,
A,G,C,T).
Support
Characteristic
Description
Inert surface
Side reactions occur less frequently because the surface of polystyrene
is inert.
Easy
derivatization
The supports are derivatized by covalently attaching the 3´-hydroxyl of
the nucleotide to the linker via a succinate ester bond, which is
base-labile and allows for removal of the DNA from the support with
ammonia.
Quantitative
cleavage
After synthesis is complete, the oligonucleotide is quantitatively cleaved,
leaving a free 3´-hydroxyl.
3-4 Automated DNA Synthesis Chemistry
Completion of the After an individual base undergoes oxidation, when other base additions are to follow,
Synthesis Cycle the synthesis cycle is completed as follows:
Phases
Description
DMT
removal
After the oxidation of a particular base, the dimethoxytrityl group is removed
with trichloroacetic acid to prepare for the next coupling.
Coupling/
Capping/
Oxidation
The previous step and these steps are repeated until chain elongation is
complete.
Concluding
phase
One of the following three options is chosen when setting up for the run, to
control the last phase:
Note
At this point, the oligonucleotide is still bound to the support with
protecting groups on the phosphates and the exocyclic amines of the bases
A, G, and C
If…
Then
the oligonucleotide
is be be purified
choose “Purify Oligo” on the Synthesis Order for the
sequence.
Note
When synthesis is complete, the DMT
group is still attached to the 5´- terminus enabling it
to act as a hydrophobic handle for purification.
the oligonucleotide
is not to be purified
choice one of the following Crude options:
♦
“Crude - DMT on” if the DMT group is not to be
removed from the sequence.
♦
“Crude - DMT off” if the DMT group is to be
removed.
Note
When making crude DNA with 5´ labels that do not have a terminal DMT (such as a
dye label), always use the “Crude-DMT on” option in order to prevent excess exposure of the
label to TCA. Always consult the instructions provided with your particular label for the correct
handling procedure.
Automated DNA Synthesis Chemistry 3-5
Overview of the Phosphoramidite Method of Synthesis
Four Chemistry A synthesis cycle is depicted below using the phosphoramidite method of
Reactions oligonucleotide synthesis. As the figure demonstrates, the synthesis cycle consists of
four chemical reactions:
♦
Detritylation
♦
Coupling
♦
Capping
♦
Oxidation
DMTO
O
B
P
support-bound
nucleoside
DMTO
O
O P
O
B
P
OCH2CH 2CN
O
O
O
B
Solid Support
DETRITYLATION
O
OXIDATION
HO
DMTO
O
B
O
B
P
P
O
O
COUPLING
P OCH 2CH 2CN
O
O
B
Add Monomer
P
DMTO
O
B
P
O
O
CAPPING
CAPPING
iPr 2N
P OCH 2CH 2CN
Phosphoramidite nucleoside
nucleotide Phosphoramidite
O
CH 3CO
O
O
3-6 Automated DNA Synthesis Chemistry
B
P
Chemistry of Choice The phosphoramidite method of oligonucleotide synthesis is the chemistry of choice
for most laboratories because it offers efficient, rapid coupling and stable reagents2.
Efficient, rapid coupling is enhanced by the following phosphoramidite chemistry
characteristics:
Characteristic
Description
Method of
derivitization
The solid support upon which synthesis begins is derivatized with the
nucleotide which becomes the 3´-hydroxyl end of the oligonucleotide.
This support material consists of beads composed of a polymer.
Support particle
and pore sizes
Support particle and pore sizes are optimized for liquid transfer and
mechanical strength.
Removal of excess
reagents
Excess reagents in the liquid phase can be removed by filtration and
washing3, eliminating the need for purification steps between cycles.
Phosphoramidite Structures and Molecular Weights
Nucleotides Phosphoramidites are chemically-modified nucleotides that are the building blocks for
synthesized oligonucleotides.
Adenine - benzoyl protected
MW 857.95
C47 H52 N7 O7 P
Thymine
MW 744.83
C40 H49 N4 O8 P
Guanine - dimethylformamidine
protected
MW 824.92
C43 H53 N8 O7 P
Cytosine - benzoyl protected
MW 833.93
C46 H52 N5 O8 P
Note
This figure shows the structures and molecular weights of standard cyanoethylphosphoramidite monomers. Exocyclic adenine (A) and cytosine (C) are protected with the benzoyl
group; the exocylic amine of guanine (G) is protected by a dimethylformamidine group (dmf).
Thymine (T) does not need a protecting group.
Automated DNA Synthesis Chemistry 3-7
Functional Groups Cyanoethyl phosphoramidite nucleotides have four functional groups
Functional Group
Description
The diisopropylamino on a 3´
trivalent phosphorous moiety4
This group stabilizes the phosphoramidite and is made
highly reactive by the activator, tetrazole.
The cyanoethyl protecting
group on the 3´ phosphorous
moiety5
This group prevents side reactions and aids in solubility of
phosphoramidites.
♦
Upon completion of the synthesis, it is removed with
ammonium hydroxide.
♦
In deprotection, ammonia acts as a base to remove a
proton on the methylene group bearing the nitrile
group.
This anion is formed only in low concentration, but rapidly
fragments by a beta-elimination reaction to form
acrylonitrile and the deprotected internucleotide
phosphodiester group. Acrylonitrile then reacts
irreversibly with ammonia to form 3-aminopropionitrile, an
inert compound.
The dimethoxytrityl (DMT, trityl)
protecting group on the 5´
hydroxyl
This group is removed during each detritylation step
leaving a reactive 5´ hydroxyl available for coupling an
incoming phosphoramidite.
The protecting groups on the
exocyclic amines of Abz, Cbz,
Gdmf (bz = benzoyl, dmf =
dimethylformadine group)
These groups prevents side reactions and are removed
upon completion of the synthesis with ammonia.
Thymidine is not protected, because it does not contain
an exocyclic amine moiety, and therefore is unreactive.
3-8 Automated DNA Synthesis Chemistry
Detritylation Chemistry Reactions
Detritylation Process Detritylation, the first step of oligonucleotide synthesis cycle, is treatment of the
derivatized solid support with the protic acid, trichloroacetic acid (TCA) in
dichloromethane, to remove the acid-labile, DMT-protecting group (as shown in the
figure below). This yields a reactive 5´ hydroxyl which can couple with a nucleotide
phosphoramidite monomer during the following coupling reaction.
Detritylation proceeds as follows:
Stage
1
Description
Immediately before detritylation, the support is washed with acetonitrile to eliminate
traces of the preceding reagent.
Note
Detritylation under anhydrous conditions is a reversible reaction. The trityl
cation is highly reactive and can re-tritylate any reactive nucleophile.
2
Detritylation is driven to completion by the removal of the trityl cation from the
synthesis/purification column.
The trityl cation is eluted by several TCA deliveries.
3
The final detritylation step is a trityl flush in which argon passes through the column
from bottom to top and pushes the liquid to a halogenated waste reservoir.
Any residual TCA is removed by an acetonitrile wash.
Depurination Purines Susceptible to Depurination
Trichloroacetic acid is a very effective protic acid detritylating agent. However, in high
concentrations of protic acids, the amine-protected purines (Abz and Gdmf) are
susceptible to depurination (removal of the purine from its sugar) via the following
pathway.6
Initial protonation at N-7 of the purine ring increases the lability of the ribose 1´-purine
N-9 bond. Cleavage of adenine and guanine bases yields a 1´ hemiacetal ribose ring,
the result of depurination. Then, during ammonium hydroxide treatment, Cleavage
occurs at the internucleotide bonds on the 3´-hydroxyl side of the apurinic
deoxyribose. (This is similar in effect to the chemistry of Maxam-Gilbert sequencing.)
Differential Exposure
Each purine in the oligonucleotide chain is exposed to acid at each detritylation step.
Purines near the 3´- end will have the longest cumulative exposure time and a greater
chance for depurination.
Automated DNA Synthesis Chemistry 3-9
Note
It has also been reported that a 5´ terminal purine is more susceptible to depurination
than an internucleotide purine.7
Quantity of DNA Cleavage Products
The quantity of DNA cleavage products generated by apurinic ammoniolysis is usually
insignificant and should not affect your product significantly.
Some things to note:
Trityl On
synthesis
If the synthesis is conducted Trityl On, for purification by OPC or trityl-on
HPLC, the 5´ end fragment of an apurinic ammoniolysis product will bear
a trityl group and may complicate purification.
Long oligos near
3´ end
Long oligonucleotides which are purine-rich near the 3´ end8
may undergo depurination.
Depurination is usually neither detectable nor significant when using ABD reagents
and cycles optimized for DNA synthesis.
To minimize depurination, each treatment with TCA should not be extended beyond
the times specified in the cycles.
IMPORTANT
3-10 Automated DNA Synthesis Chemistry
Do not stop a synthesis while the DNA is exposed to TCA.
Coupling Chemistry Reactions
Washing and Drying Before beginning the coupling step, the following steps are taken:
of Support
Step
1
Action
The support is washed extensively with acetonitrile to make it anhydrous and free of
nucleophiles (such as water).
Note
Extraneous nucleophiles compete with the support-bound 5´ hydroxyls for
the activated phosphoramidite and decrease coupling efficiency.
2
Simultaneous
Delivery of
Phosphoramidites
and Tetrazole
The support is then dried by an argon flush to remove residual acetonitrile.
According to the oligonucleotide sequence, one or more of the phosphoramidites
(Bottles 1 to 8) and tetrazole are then simultaneously delivered to the column. When
these reagents mix, the mild acid, tetrazole (pKa = 4.8), transfers a proton to the
nitrogen of the diisopropyl group on the 3´-phosphorous (see figure below).
Automated DNA Synthesis Chemistry 3-11
This protonated amine makes a very good leaving group upon nucleophilic attack by
the tetrazole to form a tetrazolyl phosphoramidite:9
Characteristic
Description
Reactive
intermediate
The tetrazolyl phosphoramidite is the reactive intermediate which forms
the internucleotide phosphite with the support-bound 5´ hydroxyl.
Excess of
tetrazole
An excess of tetrazole ensures that the phosphoramidite will be rapidly
activated.
Excess of
phosphoramidite
The excess of phosphoramidite relative to free 5´-hydroxyl ensures that
the reaction is nearly quantitative (over 98% coupling).
Mixed sequence probes are synthesized by simultaneous delivery of more than one
base (AGCT) and tetrazole with near-equivalent coupling:
Characteristic
Description
Up to four bases
Up to four bases may be specified as a mixed sequence probe. The
four nucleoside phosphoramidites have slightly different reactivities.10
Reactivity order
The cyanoethyl phosphoramidites follow the reactivity order of
T > G > C > A.
Relative
percentages
When all four are delivered simultaneously, their representation will be
(normalized to 100%):T - 30%; G - 26%; C - 24%; A - 20%.
These values are slightly dependent on the following:
♦
Cycle
♦
Location of the site in the oligonucleotide
♦
Age of the phosphoramidite solutions, and other variables.
They have a range of about 3% because of these variables.
3-12 Automated DNA Synthesis Chemistry
Capping Chemistry Reactions
Why Capping is Because coupling is not always quantitative, a small percentage of support-bound
Needed nucleotides (usually less than 2%) can fail to elongate. Such support-bound
nucleotides, however, are capable of propagating in subsequent coupling steps. As a
result, these failure sequences would contain one less nucleotide than the full-length
product and can be difficult to isolate.
Since the unreacted chains have a free 5´-OH, they can be terminated, or capped, by
acetylation with acetic anhydride and 1-methylimidazole:11
Characteristic
Description
Non-failure
sequences not
affected
The chains which reacted with the phosphoramidite in the previous
step are still blocked with the dimethoxytrityl group, so they are not
affected by capping.
Failure sequence
removed from
synthesis
Capped failure sequences do not participate in the rest of the synthesis
reactions.
Capping Process Although capping is not required for DNA synthesis, it is highly recommended
because it minimizes the length of the impurities and thus facilitates product
identification and purification (figure below).
Note
The capping reagents, acetic anhydride, and 1-methylimidazole (NMI) terminate
unreacted chains by acetylating the 5´-hydroxyl groups.
Automated DNA Synthesis Chemistry 3-13
As the figure on the previous page shows, capping proceeds as follows:
Stage
Description
1
Equal volumes and equimolar amounts of two binary reagents, acetic anhydride
and 1-methylimidazole (NMI), are delivered to the OneStep column where they mix
to create a powerful acetylating agent.
2
This agent reacts at the 5´ hydroxyls rendering these moieties unreactive for the
remainder of the synthesis.
3
The excess reagents are then removed by an argon flush (and ACN wash).
Note
Initially, the two capping reagents need to be segregated since the active acetylating
agent, acetylmethylimidazolide, is unstable. The capping time required to acetylate the 1 or 2%
of unreacted 5´ hydroxyls is very brief, only a few seconds. It is important to minimize this time
to prevent loss of cyanoethyl groups from the internucleotide linkages and to prevent base
modification by-products. Studies at Applied Biosystems have demonstrated extensively the
efficiency of both a shorter capping time and following the capping by oxidation.12
4
3-14 Automated DNA Synthesis Chemistry
Oxidation Chemistry Reactions
Oxidation Reaction After coupling, the internucleotide linkage is oxidized from the phosphite to the more
stable phosphotriester. The figure below shows the reaction that occurs when
iodine—in tetrahydrofuran, water, and pyridine—oxidizes the trivalent phosphite to the
stable pentavalent phosphate triester. This reaction is complete in less than 30
seconds.
Oxidation proceeds as follows:
Stage
Description
1
When the iodine-water-pyridine-THF mixture is delivered to the column, an
iodine-pyridine complex forms an adduct with the trivalent phosphorus.
2
When this adduct comes in contact with water, it decomposes and a pentavalent
phosphotriester internucleotide group results.
IMPORTANT
Do not stop the synthesis while the phosphorus is unoxidized.
Importance of When oxidation occurs before capping, traces of water in the oxidizing solution can
Oxidation After cause the formation of acetic acid from acetic anhydride during capping. The
Capping oligonucleotides are then exposed to acid and capping is less effective, as determined
by scientists at Applied Biosystems.
Comparison of the enzymatic digestion/base composition assay on oligonucleotides
made with different cycle orders—capping then oxidation and oxidation then
capping—shows markedly different results. When capping immediately follows
coupling11,13, a small side-reaction (the phosphitylation of the O-6 position of
guanosine, is minimized.
The Applied Biosystems standard—capping then oxidation—gives far less, usually
undetectable, amounts of base-modified nucleotides. Cycles with oxidation then
capping give high levels of the mutagenic modified nucleotide, 2,6-diaminopurine.12
Automated DNA Synthesis Chemistry 3-15
DNA Cleavage and Deprotection
Introduction When the synthesis is finished, the product and capped failure sequences are still
attached to the support as phosphate-protected, base-protected phosphotriesters.
The oligonucleotides must be cleaved from the support; complete deprotection is
necessary to produce biologically active DNA.
After Synthesis, the following occur:
Stage
Action
1
The turntable rotates, moving the OneStep column from the synthesis head to the
cleavage head.
2
Ammonia is then passed through the column, cleaving the DNA from the support.
3
After cleavage, the ammonia solution is sent to a coil heated to 65 °C for base
deprotection.
4
Deprotection of base and phosphate occur.
Cleavage Following synthesis, the DNA remains covalently attached to the support. The
oligonucleotides are cleaved from the support by treatment with fresh, concentrated
ammonium hydroxide.
As seen in the figure below, the cleavage occurs at the base-labile ester linkage
between the linker of the support and the 3´ hydroxyl of the initial nucleotide. The
cleaved DNA has a 3´ hydroxyl.
Phosphate The cyanoethyl protecting groups are removed by treatment with ammonium
Deprotection hydroxide. This occurs at the same time as cleavage, making phosphate deprotection
very quick and simple (as shown in the figure).
3-16 Automated DNA Synthesis Chemistry
Base Deprotection Base-protecting groups are removed when the DNA solution is heated to 65 °C for 1
hour in the heating coils. The deprotection process also cleaves the acetyl caps from
the failure sequences.
Base deprotection is an ammonolysis reaction, in which ammonia acts as a
nucleophile that attacks the carbonyl of the amide protecting groups. It is important to
observe these guidelines in use of ammonium hydroxide:
Guideline
Description
Fresh reagent
For effective treatment, use fresh, concentrated ammonium hydroxide on
the instrument.
Consistent‘
concentration
To ensure no decrease in ammonia concentration, store the reagent in a
refrigerator, tightly capped.
When to discard
ammonia
Discard the ammonia stock 30 days after opening.
Automated DNA Synthesis Chemistry 3-17
References
1. Efcavitch, J.W. 1988. Automated System for the Optimized Chemical Synthesis of
Oligodeoxyribonucleotides. In Macromolecular Sequencing and Synthesis,
Selected Methods and Applications. Alan R. Liss, Inc. 221-234.
2. Beaucage, S.L. and Caruthers, M.H. 1981. Tetrahedron Letters 22: 1859–1862.
3. Matteucci, M.D. and Caruthers, M.H. 1981. Tetrahedron Letters 22:1859-1862.
4. Andrus, A. and McCollum, C. 1991. Tetrahedron Letters 32:4069-4072.
Aug. 29–Sept. 2, 1989. Solid Phase Synthesis, Oxford, U.K.
Jul. 29-Aug. 3, 1990. 9th International Roundtable Nucleotides. Nucleotides &
Their Biological Applications, Uppsala, Sweden.
Applied Biosystems User Bulletin 61.
5. Sinha, N.D., Biernat, J., and Koster, H. 1983. Tetrahedron Letters 24:5843-5846.
6. Horn, T., and Urdea, M.S. 1988. Nucl. Acids Res. 16:11559-11571.
7. Tanaka, T. and Letsinger, R.L. 1982. Nucleic Acids Research 10:3249-3260.
8. Efcavitch, J.W. and Heiner, C. 1985. Nucleosides and Nucleotides 4:267.
9. Dahl, O. 1983. Phosphorus and Sulfur 18:201-204.
10. Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Summers, M.F. and Byrd, R.A. 1985.
Nucleic Acids Research 13:8181-8196.
11. Eadie, J.S. and Davidson, D.S. 1987. Nucleic Acids Research 15: 8333-8349.
Farrance, I.K., Eadie, J.S., and Ivarie, R. 1989. Nucleic Acids Research
17:1231-1245.
12. Applied Biosystems. 1988. The Effect of the Synthesis Cycle on the Chemical
Authenticity of Synthetic DNA. Research News. 239702
13. Bauer, B.F. and Holmes, W.M., Nucleic Acids Research 17, 812. 1989.
3-18 Automated DNA Synthesis Chemistry
Section 2 – Oligonucleotide Purification, Quantitation, and Storage
Topics Covered This section describes how oligonucleotides produced by the ABI™ 3948 System are
purified and quantified, and provides guidelines for storage and infomation about
alternative chemistries.
The section covers the following topics:
Topic
See page
DNA Purification
3-20
Purification Stages
3-20
Characteristics of Purification
3-20
Other Purification Methods
3-20
Overview of Oligonucleotide Quantitation
3-22
UV Spectroscopy
3-22
ODU as a Measure of Concentration
3-22
Measurement of ODU and Concentration
3-23
Introduction
3-23
UV Hardware
3-23
Setting the Baseline Absorbance
3-24
Scaling Factor
3-24
Linear Range of Absorbance
3-24
Calculation of concentration (pmol/µL) by the ABI 3948
3-25
Handling of Extinction Coefficients in Different Software
3-25
Extinction Coefficients for Fluorescent Dye Labels
3-26
Storage of the Oligonucleotide
3-27
Storage for Later Use
3-27
Storage Guidelines
3-27
Alternative Chemistries
3-28
Other Monomers
3-28
Automated DNA Synthesis Chemistry 3-19
DNA Purification
Purification Stages The ABI™ 3948 DNA synthesizer automatically performs a trityl-selective purification
of crude DNA using the polystyrene OneStep column (which has an affinity for trityl
oligonucleotides) as follows:
Stage
Description
Transfer
After deprotection, the ammonia solution of the crude trityl
oligonucleotide is transferred from the heating coils back through the
synthesis/purification column, now located at the purification head.
Note
Acetic Acid is added to the ammonia solution to neutralize it.
Retention of
oligonucleotide
The trityl oligonucleotide product is retained.
Removal of trityl
group
After water washing, the trityl group of the support-bound oligonucleotide
is removed with a mild acid solution, 3% TFA/water.
Elution of
purified
oligonucleotide
The purified oligonucleotide is then eluted with about 1 mL of a 20%
acetonitrile solution.
By-products, failure sequences not bearing a trityl group, and other
impurities are not retained and are passed out of the column.
Characteristics of The following are characteristics of purification:
Purification
Characteristic
Description
Stable to
concentrated
ammonia
The support material is stable to concentrated ammonia.
Ammonia provides
a denaturing
medium
The ammonia solution provides a denaturing medium, eliminating
secondary structure, hydrogen-bonding, and coelution of partially
complementary failure sequences.
Retention of trityl
group
The trityl group is detached and retained in the column.
Desired sample
eluted in small
volume
The purified, fully deprotected oligonucleotide is eluted in a small
volume of 20% acetonitrile in water, completely desalted and ready for
use.
Many PCR and sequencing reactions have been successfully run using DNA primers
directly from the 20% ACN/water solution.
Other Purification PAGE (polyacrylamide gel electrophoresis) and HPLC (high-performance liquid
Methods chromatography) can be used for purification but are subject to the following
qualifications:
♦
PAGE and HPLC can provide a high level of purity, but require initial capital
investment and are labor intensive and time consuming.
♦
A short oligonucleotide (<30 bases) made with typically high synthesis efficiency
(>98% average trityl yield/cycle) may require less stringent purification. Efficient
desalting and removal of non-nucleotide synthesis by-products may be sufficient
purification.
3-20 Automated DNA Synthesis Chemistry
PAGE and HPLC are elaborated in detail in Evaluating and Isolating Synthetic
Oligonucleotides, The Complete Guide, published by Applied Biosystems (Stock
Number 239902).
Automated DNA Synthesis Chemistry 3-21
Overview of Oligonucleotide Quantitation
UV Spectroscopy Nucleic acids of any variety are most easily quantified by UV spectroscopy, measuring
at or near their UV absorbance maxima, about 260 nm:
Details
Description
How measured
A dilute aqueous solution of 1 mL or less, depending on the cuvette size,
is measured by either scanning a region of about 200–350 nm or a
single 260 nm wavelength measurement.
Characteristics
of absorbance
A scan of an oligonucleotide will show broad absorbance with a maxima
near 260 nm.
ODU As a Measure A measurement of the absorbance gives a measure of the concentration of the
of Concentration solution, provided the molar extinction coefficient is known. The Optical Density Unit
(ODU) is the unit of measure of the amount of oligonucleotide.
The following definitions apply to using ODU as the Unit of Measure:
Definitions
Description
ODU and Beer’s Law
ODU
One ODU is the absorbance (typically measured at 260 nm) of a 1 mL
solution of oligonucleotide in water or an appropriate buffer at a neutral
pH range in a 1 cm pathlength cuvette.
Beer’s Law
The measurement uses Beer’s Law to allow conversion of the
absorbance reading to a molar amount. Beer’s Law states:
A = εCl, where A = Absorbance
ε = molar extinction coefficient
C = Concentration (mol/L)
l = path length (cm) typically 1 cm
Molar Extinction Coefficients
Molar extinction
coefficent
The exact molar extinction coefficient of a substance is a constant
dependent on the UV absorbing properties of the chemical structure of
that substance.
Major
contributors to
extinction
coefficient
DNA contains four major contributors to the extinction coefficient; the
four nucleobases A, G, C and T.
Each of these bases has a different extinction coefficient and a sample
of synthetic DNA has (generally) a mixture of all four.
Criteria for ODU Measurement and Conversion to Mass or Concentration
Optimum
measurement
criteria
The ODU of an oligonucleotide is generally measured at the position
when the absorbance is at a maximum (typically 260nm).
Oligonucleotides that are very rich in either purines or pyrimidines may
actually have absorbance maxima above or below 260 nm dependent on
the composition.
Criteria for
converting ODU
to mass or
concentration
Once the ODU reading is obtained, using the approximation that 1 ODU
represents about 33 µg of single stranded DNA, the mass or
concentration of an oligonucleotide can be determined provided the
molecular weight of the oligo is known.
:
3-22 Automated DNA Synthesis Chemistry
Measurement of ODU and Concentration
Introduction The ABI™ 3948 Nucleic Acid Synthesis and Purification System automatically
measures the yield of purified oligonucleotide syntheses as follows:
Stage
Action
1
Purified oligos are eluted form the purification workstation in 20% aqueous
acetonitrile and delivered to a 0.2 mm quartz flowcell within the UV station.
2
A lamp projects light at 254 nm through the flowcell and the absorbance at this
wavelength is measured.
3
The ABI 3948 System Control software converts this measurement into ODU and
picomole/mL values.
Note
The ABI™ 3948 System does not automatically determine the yield of a crude
oligonucleotide synthesis as the concentration of the DNA is too high (8-12 ODU/mL) to
determine an accurate value.
UV Hardware The ABI™ 3948 Nucleic Acid Synthesis and Purification System uses a
0.2 mL pathlength quartz flowcell to quantitate oligos. A low pressure mercury lamp
emits light at a wavelength of 254 nm to measure the absorbance of the
oligonucleotide in solution. This unit was chosen because of its compact size,
reliability and ease of maintenance.
UV lamp power
toggle switch should
be in “ON” position
UV
UV
Flowcell
Flowcell
Automated DNA Synthesis Chemistry 3-23
Lamp Lifetime The mercury lamp has an expected lifetime of one year and is replaced annually
during preventive maintenance (PN ?). If the reference voltage (baseline UV reading)
falls below 0.5 V, contact your local service engineer.
Setting the Baseline The ABI 3948 System sets a baseline absorbance value for each measurement by
Absorbance filling the flowcell with water. During operation, the most recent baseline reading is
subtracted from the oligo UV reading to give a corrected value:
ODU = (Baseline Water UV Reading – Oligo UV Reading) x 1.67
Scaling Factor The scaling factor 1.67 (or 5/3) is used because the hardware that reads the UV is
scaled to 5 volt maximum reading but the UV circuitry is designed to output to a
maximum of 3 volts.
As an example suppose the baseline water UV reading was 0.81 and the UV reading
was –1.19. The difference between the two readings is 2.00, which when multiplied by
1.67 gives a final value of 3.0 ODU.
Oligos flow into the cell either as:
♦
A solution in 20% aqueous acetonitrile, if purified, or
♦
As a solution in ammonia, if crude.
Neither of these two solutions has appreciable absorbance at the measured
wavelength.
UV Detector All UV detectors have a range of absorbance over which the response of the detector
Linearity rises linearly with increasing absorbance of the solution:
Characteristic
Description
Range of ODUs
Most measurements of ODU in laboratories are in the range 0.1 to 1.0
absorbance units.
Above or below
optimum range
Above or below this range the measured value begins to deviate from
the true value by an increasing amount
For the ABI 3948 instrument, the linear range of the UV cell is 0.5 to 3 ODU. Above
this range the response of the detector is non-linear and subject to increased error.
Therefore crude (unpurified) oligos made on the ABI 3948 instrument should be
re-quantitated for accurate measurement.
3-24 Automated DNA Synthesis Chemistry
80
70
60
50
0
40
30
20
120%
110%
100%
90%
80%
70%
60%
10
0
50%
Number of samples
The graph below provides an estimate of how close the ABI™ 3948 System can come
to approximate a bench top spectrometer. A random sample of oligos made on the
ABI™ 3948 System were taken and re-quantified on a calibrated spectrometer.
Percent Difference from true value
The graph shows that over 90% of the large sample analyzed here lies in the range ±
10%. The exceptions to these are that the sample includes some crude oligos with
ODU’s of >6.0. These have the largest error as they are outside the linear range of the
detector.
Calculation of An ODU reading is converted to pmol/µL based on the specific sequence of the oligo
Concentration made. The formula used for a sequence of length X is:
(pmol/µL) by the
10000 x ODU
pmol/µL =
ABI 3948 System
(ECbase 1 + ECbase 2 +......+ ECbase X-1 + ECbase X)
where ECbase X is the extinction coefficient of base X
For the ABI 3948 System the values for the extinction coefficient in this formula are
shown as 1% of the actual extinction coefficient for the nucleotide at each position in
the sequence as follows:
ECA = 154 ECG = 117 ECC = 73 ECT = 88
Automated DNA Synthesis Chemistry 3-25
Handling of The instrument handles extinction coefficients differently in different software, as listed
Extinction in the following table:
Coefficients in
Description
Different Software Software Version
2.0 (NPR)
Extinction coefficients of bottles 5–8 are taken as an average of
the above four values i.e. )
(154 + 117 + 73 + 88)
= 108
4
These values are hard-wired into the image and are not
editable.
2.20 (Post-NPR)
Values for A, G, C and T are hard-wired into the image but
bottles 5-8 are operator specified in the Instrument Preference
Page to allow for different 5´labels having different extinction
coefficients.
Extinction The relevant extinction coefficients for the available Fluorescent dye labels are
Coefficients for ♦ 6-Fam - 18363
Fluorescent Dye
Labels ♦ Hex - 28207
♦
Tet - 14642
As an example, the 20 base sequence 5’ ATC TAC CTT GGC TGT CAT TG 3’ has
three As, four Gs, five Cs and eight Ts. The sum of the extinction coefficients is
therefore:
3 x ECA + 4 x ECG + 5 x ECC + ECT = 462 + 468 + 365 + 704 = 1999
If the reported ODU for this oligo was 3.1 then the pmol/µL would be:
31,000
1999
3-26 Automated DNA Synthesis Chemistry
= 15.5 pmol/µL
Storage of the Oligonucleotide
Storage for Later Most applications for synthetic oligonucleotides require less DNA than the typical 0.5
Use to 4 ODU of purified oligonucleotide that the ABI 3948 System produces. Fortunately,
oligonucleotides can be stored easily, with little or no degradation for long periods of
time.
Storage Guidelines Use the following guidelines for storage:
Guideline
Description
Storage
refrigerated in
solution
It is most convenient to store them refrigerated as a solution, in either
a crude or purified state.
Storage frozen in
20% acetonitrile
Purified oligonucleotides may be stored frozen in the 20% acetonitrile
solution.
Storage of crudes
Oligonucleotides synthesized and collected crude may be stored in
the concentrated ammonia solution.
Other liquid
storage media
Other storage media include:
Dry storage
♦
Ethanol
♦
Acetonitrile
♦
Triethylammonium acetate (TEAA)
♦
Ethylenediamine tetraacetic acid (EDTA)
Alternatively, oligonucleotides may be stored as a dried pellet in a
clean, dry vessel such as a micro-centrifuge tube.
Note
Except for oligonucleotides in dry storage, keep oligonucleotides cold to minimize
degradation and bacterial growth.
When stored by the above means, oligonucleotides are stable for over a year. Avoid
storing oligonucleotides in solutions that are mutagenic, oxidizing, or outside the pH
range of 3–12.
Automated DNA Synthesis Chemistry 3-27
Alternative Chemistries
Other Monomers In addition to synthesis with the standard phosphoramidite monomers, other
monomers may be used on the ABI 3948 System synthesizer. These other monomers
include:
♦
Aminolink
♦
Biotin
♦
Deoxyinosine
♦
Phosphalink, and the
♦
Fluorescent amidites
Note
For more information on the monomers and alternate chemistries listed above, visit the
Applied Biosystems Nucleic Synthesis web page as described below.
Step
Action
1
In your web browser, enter www.appliedbiosystems.com/techsupport as the URL
and click "Open" or otherwise enable the web connection to the Applied Biosystems
Products and Applications web page.
2
In the Products and Applications web page, click the "Nucleic Acid Synthesis"
button.
3
In the Nucleic Acid Synthesis web page, connect with the web page for the
chemistry reagent or other topic of interest by clicking the appropriate button.
Please note the following qualifications when using other monomers:
♦
When using Aminolink or Phosphalink, select the “Crude, DMT-Off” post synthesis
option in the New Synthesis Order screen.
♦
When using the fluorescent amidites, use the “Syn-Pure x.xx Dyes” run protocol.
♦
When usinging Biotin, select the “Syn-Pure x.xx Biotin” run protocol.
Note
The “x.xx” presented in the names above represents the version numbers of Dye and
Biotin protocols on your instrument, which vary with date of purchase/software release.
3-28 Automated DNA Synthesis Chemistry
Overview of Software
Commands
4
4
In This Chapter
Topics Covered This section provides information on the command menus available from the Menu
bar. A general knowledge of these commands is required to operate the ABI 3948
System Control application. In addition, the New Synthesis Order command contains
information about the Synthesis Order form.
This chapter contains three sections:
Section
See Page
File and Edit Menus
4-2
Synthesizer and Window Menus
4-20
RunFiles, Sample Labeling, Synthesis Order and Multi-Order Files
4-31
Overview of Software Commands 4-1
Section 1 – File and Edit Menus
Topics Covered This section describes the commands on the File and Edit menus and covers the
following topics:
Topic
Overview of File Menu Commands
See page
4-3
Menu Commands
4-3
Command-Key Commands
4-4
New Synthesis Order Command
4-5
Introduction
4-5
Types of Information
4-5
Description of Fields
4-5
Sequence Entry
4-6
Open Command
4-8
Introduction
4-8
Synthesizer Windows
4-8
Synthesis Orders
4-8
RunFiles
4-8
Multi-Order Files
4-9
Open Synthesizer Command
4-10
Dialog Box
4-10
Procedure
4-10
List of Synthesizer Window Views
4-11
Other File Menu Commands
4-13
Send to Synthesizer Command
4-13
Save a Copy In Command
4-13
Send Copy to Synthesizer Command
4-14
Save and Reopen Command
4-14
Import/Export Command
4-15
Export Procedure Icons
4-16
Generate Run File Command
4-16
Overview of Edit Menu Commands
4-17
Read Selection Command
4-17
Find Command
4-18
Find Same Command
4-18
Replace Command
4-18
Replace Same Command
4-19
Use Sounds Command
4-19
4-2 Overview of Software Commands
Overview of File Menu Commands
Menu Commands The File menu contains commands to perform the following tasks:
File
Command
Description
New Synthesizer Order
Creates a new Synthesis Order.
Open
Opens a saved Synthesis Order or saved Synthesizer
database file.
Opens a Run File for label printing.
Opens a text file to produce Multi-order files.
Open Synthesizer
Establishes communication with a synthesizer.
Send to Synthesizer
Sends all synthesis setup information from a Synthesizer
window to a synthesizer.
Save a Copy In
Saves a copy of all information from a Synthesizer to a
Synthesizer database.
Send Copy to
Synthesizer
Sends a database copy of synthesis setup information to a
synthesizer.
Close
Closes the selected open Synthesizer window, Synthesizer
database, or Synthesis Order,
Close All Synth Orders
Closes one or more open Synthesis Orders,
Save
Saves changes made to a selected Synthesizer window,
Synthesizer database, or Synthesis Order (Save).
CAUTION Be aware that using the Save command with a
Synthesizer window to which you have made changes
downloads those changes to the synthesizer. You may not
want to do this. It is preferable to use the Save As
command first, do your edits on the copy, and then use the
Send Copy to Synthesizer command.
Using Save with a Synthesizer database or Synthesis Order
saves the changes to a Macintosh file.
Save As
Saves changes made to an active window to a new file,
enabling the file to be renamed.
Save & Reopen
Used to fill out multiple orders for the same customer.
Import Sequence
Used to import cycles/procedures/sequences created in
another application to an open Synthesis Order or Synthesizer
window.
Export Sequenceb
Used to export cycles/procedures/sequences from an open
Synthesis Order or Synthesizer window.
Generate Run File
Generates a Run File for the synthesis.
Page Setup
These are regular Macintosh® commands and are not
described here.
a
Print
Quit
If you need information on using standard Apple commands,
see the Macintosh System Software User’s Guide that came
with your computer
a. This command reads Import Cycle for an Edit Cycle view or Import Procedure for an Edit Procedure view.
b. This command reads Export Cycle for an Edit Cycle view or Export Procedure for an Edit Procedure view.
Overview of Software Commands 4-3
Note
If you have logged on to the synthesizer using a Monitor Access password, the only
commands available are Open Synthesizer, Close, and Quit.
Command-Key As an alternative for the mouse in choosing some commands, you can use the
Commands Command-key equivalent that appears next to some commands on the pop-up menu.
For example, to choose the Open command, hold down the Command () key and
press O.
Note
If you have logged on to the synthesizer using a Monitor Access password, the only
commands available are Open Synthesizer, Close, and Quit.
4-4 Overview of Software Commands
New Synthesis Order Command
Introduction This command opens an empty Synthesis Order like that shown below.
Types of Information The New Synthesis Order command is used to document synthesis order information
so that it can be transferred to the synthesizer. Synthesis information includes:
♦
Sequence listing
♦
Names of cycles
♦
End procedure
♦
DMT state used for synthesis
Note
The Synthesizer Order window (below) automatically displays information in the Order
Date field.
Description of Fields The fields in the Synthesis Order are described in the table below.
These are the fields available from the File Menu:
Field Name
Description
Customer Name
A field to identify the customer.
Customer Address
A field for the customer’s address.
Phone /Fax #
A field for the customer’s phone and/or Fax numbers.
PO Reference
A field for the customer’s purchase order number.
Accnt#
The account number assigned to the customer by ABD.
Order Date
The date the customer placed the New Synthesis Order. This date
corresponds to the date shown on the computer’s General Controls
in the Control Panel.
Comments
Any information needed to further describe the sequence.
Note
Entries in this field should be limited to 49 characters since
only the first 49 characters are transferred to the RunFile or
Overview of Software Commands 4-5
These are the fields available from the File Menu: (continued)
Field Name
Description
Protocol Options
Two pull-down menus are available on this line of the Synthesis
Order form.
♦
The first menu allows you to assign any of 15 pre-defined
protocols for production of the sequence.
♦
The second pull-down menu allows the following three choices
for the oligonucleotide.
–
Purify Oligo - When this choice (the default) is selected, the
synthesized oligo is automatically purified.
–
Crude - DMT Off - When this choice is selected, the
oligonucleotide is not purified and the DMT group at the 5'
end of the sequence is automatically removed.
–
Crude - DMT On - When this choice is selected, the
oligonucleotide is not purified and the DMT group at the 5'
end of the sequence is left on.
Sequence Name
This field allows you to enter up to 31 characters for the sequence
name.
Sequence
This field is where you enter the component oligonucleotides in the
sequence. Besides the four bases, valid entries include 5, 6, 7, and 8
(bottle positions), and single-character IUB ambiguity codes.
Sequence Entry As you enter a sequence in the Sequence field, the number of A, G, C, T bases are
counted in the bottom of the Synthesis Order form and the length of the sequence is
listed in the left-hand column. The IUB ambiguity codes are counted as one base for
the length of the oligonucleotide.
After you have entered a sequence, you can check your typed entry against a hard
copy as follows.
To check your sequence entry:
Step
Action
1
Highlight the entry in the window.
2
Choose the Read Selection command from the Edit menu. A synthesized human
voice reads the sequence back to you.
When the Synthesis Order form is completed, you can save the order either as:
♦
A Synthesis Order (Save and Save As commands), or
♦
As a sequence file (Export Sequence command)
Synthesis Orders are used both as a record of a customer order and to input the
sequence and other information needed for the instrument to produce the
oligonucleotide.
4-6 Overview of Software Commands
Note
Be sure to end the sequence on the 3´ end with A, B, C, or T or you will be presented
with the message shown in the figure below (synthesis starts at the 3´ end of the sequence and
only A, C,G, or T are permissible entries).
When a Synthesis Order is opened in Microsoft Word or other word processors, it
contains a complete record of the information entered into the sequence order. When
a Synthesis Order is saved as a sequence file and then opened in Word (or other
software), it contains only a listing of the sequence.
Overview of Software Commands 4-7
Open Command
Introduction This command opens a directory dialog box (figure below) that displays files created
by the ABI 3948 System Control application and stored on the hard disk or on a floppy
disk.
The types of files which may be opened by this command include the following:
♦
Synthesizer Window or database files
♦
Synthesis Order files
♦
RunFiles
♦
Multi-Order text files
Synthesizer A Synthesizer Window is the window used to control the ABI™ 3948 DNA Synthesis
Windows and Purification System. Complete information on the window is provided in Chapter 5
of this manual.
Synthesis Orders Synthesis Orders are the documents loaded into the Synthesizer Window to produce
oligonucleotides. Information on this document is presented in several places in the
ABI™ 3948 System User and Reference Manuals:
♦
Information about Synthesis Orders is provided under ““New Synthesis Order
Command” on page 4-5 and under “About Synthesis Orders” on page 4-38.
♦
Information on preparing individual Synthesis Orders is provided under “Preparing
Synthesis Orders” on page 4-8 of the ABI™ 3948 System User’s Manual.
♦
Information on the text files used to create multiple Synthesis Orders is contained
in Appendix C of the ABI™ 3948 System User’s Manual.
RunFiles Information on RunFiles is provided in Section 3 of this chapter. Since RunFiles are
used as a source for label information, Section 3 also provides the following
information to support the process of label generation:
♦
Detailed information on the contents of RunFiles is provided under “RunFiles” on
page 4-32.
♦
Information on generating labels using the ABI 3948 System Control application is
provided under “Sample Labeling Feature” on page 4-33.
♦
Detailed information on the ABI 3948 System Control window used to generate
labels is provided under “Label View Information” on page 4-36.
4-8 Overview of Software Commands
Multi-Order Files Information on Multi-Order files, which are text files used to produce multiple
Synthesis Orders, is provided under“Multi-Order Files” on page 4-41 in this section.
For detailed information on the syntax of such files, see Appendix C of the ABI™ 3948
System User’s Manual, “Creating and Using Multi-Order Files.”
Overview of Software Commands 4-9
Open Synthesizer Command
Dialog Box Establishes communication with an instrument on either a single zone or multi-zone
network. When you choose this command, the Open Synthesizer Dialog box (below)
opens.
This dialog box can display a multi-zone network, or list synthesizers on a single zone
network.
Note
The default application memory setting is 3048K. You can have a maximum of four
databases and 48 orders open. If you need to open more windows, you will need to assign more
memory to the ABI 3948 System Control application by selecting the application icon
(application closed) and using Command-I.
Procedure To establish communication with a synthesizer:
Step
1
Action
Choose Open Synthesizer in the File menu.
2
Select the zone name in the scroll box labeled Apple Talk Zones, if applicable.
3
Select a synthesizer in the box labeled Select a Synthesizer.
4
Click OK to present a password dialog box.
5
Enter a password and click OK.
A Synthesizer window like that shown on page 4-11 appears.
4-10 Overview of Software Commands
List of Synthesizer A pop-up menu at the top of the Communication View, the initial Synthesizer window,
Window Views displays the views listed below the figure.
x.xxx
The Synthesizer window contains the following views which provide the options you
need to define your synthesis run, monitor the run, manually control the run and edit
cycles and procedures:
Name of View
Purpose
Communication
This is the initial view shown above.
Run Setup
This view is used to set up a run.
Run Protocol
This view is used to define the chemistry protocol to
be used for a run.
Monitor Chemistry
This view is used to monitor chemistry in progress
during a run.
Monitor Instrument
This view is used to monitor instrument hardware
during a run.
Monitor Run
This view is used to monitor the production of
oligonucleotides during a run.
Edit Synthesis Cycle
This view is used to create and edit synthesis cycles.
Edit Cleavage Cycle
This view is used to create and edit cleavage and
deprotection cycles.
Edit Purification Cycle
This view is used to create and edit purification cycles.
Edit Begin Procedure
This view is used to create and edit begin procedures.
Edit End Procedure
This view is used to create and edit end procedures
Edit Bottle Procedure
This view is used to create and edit bottle procedures.
Misc (Miscellaneous) Procedures
This view is used to create and edit various types of
procedures.
Manual Control
This view is used to exercise manual control over the
instrument.
Power Fail History
This view reports power failures which occur on the
instrument.
Overview of Software Commands 4-11
Name of View
Purpose
Instrument Test
This view is used to test instrument hardware.
B+Tet Calibration Table
This view displays values used to provide maximum
times for phosphoramidite deliveries.
Reagent Utilization Table
This view is used to calculate the amounts of reagents
required during run setup.
When you choose one of these views from the pop-up menu, the contents of the
Synthesizer window change to display the related options.
Chapters 5 and 6 provide complete descriptions of all the views in the Synthesizer
window and the available options.
4-12 Overview of Software Commands
Other File Menu Commands
Send to Synthesizer This command sends the contents of the Synthesizer window to a synthesizer. Use
Command the command to send information to the ABI™ 3948 Nucleic Acid Synthesis and
Purification System after creating new or modified versions of the following:
♦
Run Protocols
♦
Cycles - synthesis, cleavage/deprotection, or purification cycles.
♦
Begin, End, Bottle, or Shutdown procedures.
♦
Functions
♦
Time/Date setting
Note
If you have made changes to a Synthesizer window and intend to save them, you
should use the Save a Copy In command before using the Send to Synthesizer command,
because this is the most direct way of saving your changes.
Save a Copy In This command makes a complete copy of the run setup, protocols and all other
Command cycles, procedures, and settings for the ABI™ 3948 Nucleic Acid Synthesis and
Purification System. It places the information in a computer file, which serves as a
backup.
When you choose this command, an alert box like that above informs you that the
copying process takes some time.
IMPORTANT
database.
The Save a Copy In command copies the contents of the Synthesizer
When you click OK to initiate saving a copy, a directory dialog box like that shown
below appears.
Assign the file name and location in the directory dialog box.
Overview of Software Commands 4-13
Send Copy to This command sends a database file that contains all the information in an open
Synthesizer database window to the ABI™ 3948 Nucleic Acid Synthesis and Purification System.
Command The database file is created when you choose Save a Copy In... (see page 4-13).
This command is useful for:
♦
Transferring saved synthesizer information from one instrument to another
instrument on the network.
♦
Restoring all information and instrument settings to a previous backup version.
The combination of Save a Copy In and Send Copy to Synthesizer allows many
users to share one synthesizer and still have completely individual customized
setups.
When you choose this command, a dialog box (see below) displays the ABI™ 3948
instruments that are connected to the computer.
Synthesizer-1
Note
For multi-zone networks, the dialog box displays a zone list.
IMPORTANT
You will need to recalibrate the sensors only when you reset the database.
Sending a copy of a new database does not require a sensor calibration.
Save and Reopen This command is:
Command ♦ Useful when filling out a number of Synthesis Orders for the same customer.
♦
Allows you to enter another sequence without reentering the other information on
the form (as you would need to do if you used the New Synthesis Order form).
Note
This command is like the Save and Save As commands except that the version of the
Order remaining open does not contain any of the sequence information just saved.
4-14 Overview of Software Commands
Import/Export The Import and Export commands are available when you are creating a Synthesis
Command Order or creating and editing cycles and procedures in a Synthesizer window. The
command name changes to reflect the name of the capability that is currently active,
as described in the table below:
Command
Where available
Import Sequence
Synthesis Order
Import Cycle
Edit Synthesis Cycle
Edit Cleavage Cycle
Edit Purification Cycle
Import Procedure
Edit Begin Procedure
Edit End Procedure
Edit Bottle Procedure
Misc Procedure
Export Sequence
Synthesis Order
Export Cycle
Edit Synthesis Cycle
Edit Cleavage Cycle
Edit Purification Cycle
Export Procedure
Edit Begin Procedure
Edit End Procedure
Edit Bottle Procedure
Misc Procedure
Export Procedure The Export Procedure command produces a specific type of file. Once exported, you
Icons can import a file type into any window that corresponds to the file type. The various file
types and corresponding icons are shown in the figure below.
Sequence file or
Synthesis Order icon
Begin Procedure
file icon
Cycle file icon
End Procedure
file icon
Bottle Procedure
file icon
Miscellaneous Procedure
file icon
Overview of Software Commands 4-15
Generate Run File Although a RunFile (see below) is normally generated automatically at the end of a
Command run, you are always prompted by the dialog box shown in the right side of the figure
when you click the Load button in the Run Setup view.
The type of RunFile saved by clicking Yes in the dialog box is a TRunFile, the same
type produced by the Generate Run File command (the keyboard shortcut for the
command is Command - M).
4-16 Overview of Software Commands
Overview of Edit Menu Commands
Overview of Edit The Edit menu commands perform the following tasks:
Commands
Command
Description
Undo Typing/Redo Typing
Standard Apple Edit Menu Commands.
Cut
Note
If you need information about these commands, see
the Macintosh System Software Users Guide that came with
the computer.
Copy
Paste
Clear
Read Selection
Reads a sequence or portion of a sequence in the Edit
Sequence window out loud (Read Selection).
Find/Find Same
Find (or find again) a particular sequence of bases in the Edit
Sequence view.
Replace/Replace Same
Replace (or replace again) a particular sequence of bases in
the Edit Sequence view with a designated sequence of bases
Select All
Standard Apple Edit Menu Command (see Note above).
Use Sounds
Turn on or turn off the sounds that accompany communication
between the computer and the 3948 (Use Sounds).
Show Clipboard
Standard Apple Edit Menu Command (see Note above).
Besides choosing a command from a menu using the mouse, you can choose a
command by using the Command key equivalent that appears on the menu next to the
command. For example, choose the Read Selection command by holding down the key and then pressing K.
Note
If you have logged on to the synthesizer using a Monitor Access password, only the
Open Synthesizer, Close, and Quit commands are available.
Read Selection This command reads aloud the sequence entered into the New Synthesis Order. You
Command can check the entered sequence by comparing the vocalized sequence to the original
sequence.
To hear the sequence read aloud:
Step
Action
1
Click the cursor on the sequence.
2
Use the Select All command to highlight the entire sequence, or drag the cursor
across the sequence to highlight a portion of the sequence.
3
Choose Read Selection. A synthesized voice reads the order of bases entered in
the Sequence field.
4
To interrupt the reading, press the Shift key. The reading stops and unread text
remains selected.
Overview of Software Commands 4-17
Find Command This command locates a particular sequence of bases in an Synthesis Order.
To find a defined sequence:
Step
Action
1
Click the cursor in the sequence.
2
Choose the Find command. A dialog box like that below appears.
3
Enter the pattern you want to find in the dialog box and click Find.
If the pattern is in the sequence, it is highlighted. If the pattern is not found,
the computer emits a beep.
Note
As a convenience, both Find and Replace ignore differences between
lower and upper case letters. You may type everything in lower case. 3948 Control
software automatically converts the text to upper case.
Find Same This command locates the second and subsequent occurrences of the pattern
Command specified by the Find command.
Replace Command This command finds one pattern of bases and replaces it with another.
To replace one pattern of bases with another:
Step
Action
1
Click the cursor in the sequence.
2
Choose the Replace command. A dialog box like that below appears.
3
Enter the pattern you want to remove in the field labeled Find what string?
4
Enter the sequence you want to substitute in the field labeled Replace with what
string?
5
Click Replace to change only one occurrence of the base pattern.
Click Replace All to change all occurrences of the base pattern.
Click Find to locate each occurrence of the base pattern.
4-18 Overview of Software Commands
Replace Same This command repeats the substitution designated in the Replace dialog box (see
Command figure above).
Use Sounds This command controls the built-in sounds that accompany communication on the
Command network. The command acts like a toggle switch, alternating between the off and on
positions.
The sounds verify that communication is occurring (and you may find them
entertaining). However, the sounds use computer memory and can slow down
communication. Communication is noticeably faster with the sound turned off.
Overview of Software Commands 4-19
Section 2 – Synthesizer and Window Menus
Topics Covered This section describes the commands on the Synthesizer menu, the contents and
function of the Instrument Preferences window, and the purpose of the Window menu.
The section covers the following topics:
Topic
Overview of Synthesizer Commands
Menu Commands
Abort Command
Procedure
See Page
4-21
4-21
4-22
4-22
Interrupt Command
4-22
Description
4-22
Resume Command
4-22
Description
4-22
Pause After Command
4-23
Introduction
4-23
Programming a Pause
4-23
Synchronize Clocks Command
4-24
Procedure
Change Password Command
Procedure
Change Name Command
Procedure
4-24
4-25
4-25
4-26
4-26
Instrument Preferences Command
4-27
Purpose of the Command
4-27
Auto-Resume Feature
4-27
Setup Variables
4-28
Setup Choices
4-29
Instrument Dip Switches
4-29
Window Menu
Description
4-20 Overview of Software Commands
4-30
4-30
Overview of Synthesizer Commands
Menu Commands The Synthesizer menu provides the commands listed in the table below:
Synthesizer
Command
Description
Abort
This command terminates a run in progress.
CAUTION
Interrupt
Aborted runs can not be resumed.
This command interrupts a run in progress. Use this command
when you intend to resume the run.
Note
Relay 2 is turned on when a run is Interrupted, is
turned off when the run is Resumed, and is pulsed for
two-tenths of a second when a run ends. This is done to allow
relay 2 to trigger an alarm or other device to alert the operator
when a run has paused or ended.
Resume
This command resumes an interrupted run.
Pause After
This command is used to program a pause during a run. Runs
halted with this command are started with the Start button, not
the Resume command.
Synchronize Clocks
This command is used to synchronize the instrument’s clock
with the computer’s clock.
Change Passwords
This command is used to change the passwords assigned to
the instrument.
Change Name
This command is used to change the name assigned to the
instrument.
Instrument Preferences
This command is important in setting up a run because it opens
a special window used to control a number of instrument
parameters.
Note
These commands are not available if you have logged on to the synthesizer using a
Monitor Access password.
Overview of Software Commands 4-21
Abort Command
Procedure This command stops synthesis immediately. After you use the Abort command, you
must completely start over, setting up your run again using the Run Setup window.
The Abort command is used as follows:
Step
1
Action
Choose Abort from the Synthesizer pop-up menu while a run is in progress. This
will present the following dialog box:
IMPORTANT
Be certain that you really want to abort the current synthesis
since this command stops all active columns and you must completely start over
again.
2
Click Yes if you want to abort the current run or click No if you have reconsidered.
Interrupt Command
Description The Interrupt command halts synthesis at the next Safe step and also disables Auto
Resume. You can re-start synthesis after an interrupt with the Resume command.
Note
A Manual Control action can not be interrupted but procedures can.
Relay 2 is turned on when a run is Interrupted, is turned off when the run is Resumed,
and is pulsed for two-tenths of a second when a run ends. This is done to allow relay 2
to trigger an alarm or other device to alert the operator when a run has paused or
ended.
Resume Command
Description This command re-starts a run after an interrupt.
4-22 Overview of Software Commands
Pause After Command
Introduction When you choose the Pause After command (Synthesizer menu) while a run is in
progress, a dialog box like this is presented.
The Pause After command enables you to:
♦
Pause a run after any synthesis listed (and still not completed) in the dialog box.
In the figure example above, only one choice is possible for setting a pause after.
♦
Set a pause after a number of listed syntheses, if you use the command earlier in
the run.
Programming a A Pause After has the following characteristics:
Pause
Characteristic
Description
Execution after
end of
synthesis
A Pause After is executed only after the end of synthesis.
Information in
dialog box
Besides indicating where a run may be paused, the dialog box provides
the following information:
This is necessary because a turntable move is needed to load new
columns and the synthesis jaws must remain sealed until synthesis
chemistry is completed
♦
Syntheses completed (marked by *1)
♦
Synthesis currently in progress (marked by *4)
♦
Whether a synthesis is scheduled (marked by *2) or not scheduled
(marked by *3).
Set All button
The Set All button allows you to set a pause after every available position
Clear All button
The Clear All button clears any choices you have made.
When you are ready to save changes to your pause choices, do the following:
♦
Click the Save Pause Options button.
♦
After the system pauses at the programmed point, extend the current run as
described in the User’s Manual in “Extending a Run” on page 2-35 of the ABI 3948
User’s Manual.
Overview of Software Commands 4-23
Synchronize Clocks Command
Procedure This command resets the clock on the ABI™ 3948 instrument to match the clock on
the computer. When you choose the command, a dialog box like that below appears.
8
8
To synchronize the synthesizer and computer clocks:
Step
Action
1
Choose the Synchronize Clocks command in the Synthesizer menu.
2
Check the date and time that appears after the word Macintosh.
If it is not correct, click Cancel to close the dialog box. Reset the current date and
time on the computer’s Control Panel. See the Macintosh System Software User’s
Guide for instructions.
3
T
4-24 Overview of Software Commands
When the Macintosh time and date are correct, click OK in the dialog box. The
synthesizer’s time and date settings change to match the Macintosh settings.
Change Password Command
Procedure This command is used to assign or change a password.
To assign or change a password:
Step
1
Action
Choose the Change Password command from the Synthesizer menu.
This brings up the dialog box to the left below.
2
Do one of the following:
♦
If no password has been assigned yet, click OK without entering a password.
♦
Enter the Full Access password if one is assigned.
Either of these actions presents the dialog box shown below:
Note
If you enter the wrong password when one has previously been assigned,
the dialog box shown to the right in step 1 appears.
3
Do one of the following:
♦
For Full Access, click the Full Access radio button and then click the Continue
button.
♦
For Monitor Access, click the Monitor Access radio button and then click the
Continue button.
Either of these actions will present the dialog box shown to the left below.
4
Re-enter the password you entered in step 2. If you fail to enter the same password,
you will be presented with the dialog box shown to the right above.
Overview of Software Commands 4-25
Change Name Command
Procedure This command assigns a name to a synthesizer. The Synthesizer name appears when
you choose the Open Synthesizer command to establish communication with the
synthesizer, and in the title bar of the Synthesizer window.
When you choose the Change Name command, a dialog box appears like that shown
below.
Do the following to change a name:
Step
Action
1
Type a synthesizer name in the entry field labeled Name. The entry field holds up to
32 characters.
2
Click OK to finish assigning a name.
4-26 Overview of Software Commands
Instrument Preferences Command
Purpose of the The command is used to present the Instrument Preferences window, shown below.
Command This window allows a number of chemistry and hardware settings to be made in one
place:
Type of
Setting
Description
Setup
Variables
Setup Variables allow the user to change values for key functions in
chemistry cycles.
Setup Choices
Setup Choices allow a number of choices for hardware or operation
conditions.
Instrument Dip
Switches
Instrument Dip Switches show the settings of dip switches on the
processor (CPU) board and should only be changed by an ABD Service
Engineer.
The ability to make a number of changes in one place facilitates adjusting the
instrument to the specific environmental conditions found in different labs. The
Instrument Preferences window also is intended to support the Auto-Resume
instrument feature.
Auto-Resume The Auto-resume feature, described under “Auto-Resuming” on page 2-17 of the ABI
Feature 3948 System User’s Manual, is intended to strike a balance between throughput and
reliable operation. See “Auto-Resuming” in the referenced material for more
information on using the Auto-resume feature.
65
60
33
380
35
200
15
130
Overview of Software Commands 4-27
Setup Variables Entries made for Setup Variables directly change parameter values of a number of key
functions in chemistry cycles. This capability allows the user to configure the
instrument for different operational conditions. The values shown in the Instrument
Preferences example on page 4-27 are the default values and can be changed by
selecting the existing entry and typing in a new value.
Note
Values entered for Setup Variables in this window are default values: entering a
“non-zero” value into the Misc field of the corresponding step of the associated cycle
supersedes the Instrument Preferences setting.
Functions affected include:
Function or
Parameter
Description
Deprotect Temp (170)
This variable (for Function 170, “Start Depro Htr”) sets the final
temperature at which the deprotection coils are set for
deprotection. Final temperature is set here rather than by the
associated step in the Cleavage cycle.
Deprotect Mins (169)
This variable (for Function 169, “Depro Htr Wait”) sets the minimum
time that the deprotection coils will remain hot once the final
deprotection temperature has been reached. This time is set here
rather than by the associated step in the purification cycle.
Xfer Into Coil (260)
This variable (for Function 260, “Set Coil Temp”) sets the
temperature at which the deprotection coils are set to for the
transfer of the sample from the cleavage vessels into the
Deprotection coils. This temperature is set here rather than by the
associated step in the purification cycle.
Xfer From Coil (261)/
Coil Cool Secs (261)
These variables (for Function 261, “Wait Coil Temp”) sets the target
temperature for the deprotection coil and the time allowed for the
temperature to be reached before the transfer from the coils to the
purification columns. Temperature and time are set here rather
than by the associated step in the purification cycle.
Jaw Leak Test in PSI
This variable sets the pressure used for the Jaw/block leak test that
is executed when the jaws close after each turntable move at the
beginning of each cycle.
Leak OK 0.01 PSI
This is the maximum passing leak rate for the Jaw/Block Leak Test
in 1/100 of PSI.
Auto-resume Minutes
This is the time in minutes that the system waits before
auto-resuming from a failed sensor delivery.
Auto-res OK 0.01 PSI
This is the maximum jaw leak in 1/100 PSI allowed to keep
auto-resume enabled - may not be as large as the Leak OK value.
Ext. Coefficient 5
The values for these parameters represent 1% of the extinction
coefficient values for the contents of Bottles 5-8. These values are
used to convert ODU values to pmole/mL.
Ext. Coefficient 6
Ext. Coefficient 7
Ext. Coefficient 8
4-28 Overview of Software Commands
These values can be adjusted to give more accurate quantitation
when using bottles 5–8 (default = 10800).
Setup Choices These checkboxes provide a central place to make the following hardware or
operational settings.
Setup Choice
Description
User 4x12 Tube Rack
Check this box when you want to use the white rack (4X12
configuration). Leave the box blank to use the red rack (8X6
configuration). This box is unchecked by default.
Pause On Sensor Fail
The instrument will pause on a failed sensor delivery with this box
checked. The box is checked by default. The system may
auto-resume from this pause if auto-resume is enabled.
Pause On Jaw Leak
The instrument will pause on a failed leak test with the box
checked (unchecked by default). The system will not auto-resume
from this pause.
End Row on Jaw Leak
The affected turntable row will drop out of the run on a failed jaw
leak test but the run will continue processing other rows. This box
is checked by default.
Man Cont Jaw Testing
This parameter enables jaw/block testing in Manual Control mode.
This box is unchecked by default.
Log Dry Sensor Fxns
This parameter enables “dry” sensor flows (flushes and
backflushes) report to the Microphone log - normally only “wet”
sensor deliveries report. Default is unchecked.
Note
Be aware that enabling this parameter will produce a
much larger Microphone file.
Mnfg. Jaw Test Mode
Only used during instrument manufacturing.
Instrument Dip These are hand Dip switches on the CPU board whose settings are reflected on this
Switches page. They may only be set by taking off a side panel and flipping the switches on the
board.
Dip Switch
Description
Check Illegal Values
This parameter is enabled by default. It makes sure no “illegal”
value combinations are being attempted (especially by User
functions).
No Flow To Open Jaws
This box is checked by default since this function is only used to
test deliveries to the jaws.
Reserved
Reserved for future use.
Overview of Software Commands 4-29
Window Menu
Description The Window menu displays all open Synthesis Orders, Database, Synthesizer, and
Print Label windows. When you can select one of the items listed, that window moves
to the front of the monitor display and becomes the active window.
Window
Note
A Print Label window is a window opened by opening a RunFile window in the ABI™
3948 System Control application. Information presented for such files includes date of creation
(in month, day, year) and time of creation (hour, minute, second, AM or PM). For more
information on RunFiles, see “RunFiles” on page 4-32.
4-30 Overview of Software Commands
Section 3 – RunFiles, Sample Labeling, Synthesis Order and Multi-Order Files
Topics Covered This section describes RunFiles, how to use RunFiles to label samples, and Synthesis
and Multi-Order files.
The section covers the following topics:
Topic
RunFiles
Information in the RunFile
Sample Labeling Feature
Procedure
Label View Information
See Page
4-32
4-32
4-33
4-33
4-36
Introduction
4-36
Information in the Five Columns
4-36
Information in Individual labels
4-36
Label Preferences Button
About Synthesis Orders
4-37
4-38
Introduction
4-38
Ways of Creating Synthesis Orders
4-38
Synthesis Order Sequence Entry
4-39
Importing and Exporting Sequences
4-39
Actions after Sequence Entry
4-40
Multi-Order Files
4-41
Introduction
4-41
Applications Used to Create
4-41
Generating Synthesis Orders
4-43
Overview of Software Commands 4-31
RunFiles
Information in the The ABI 3948 System Control application generates a RunFile for each run. Run
RunFile information is normally automatically generated and stored in a file like that shown
below. The information on which a RunFile is based is no longer available from the
instrument after the start of the next run.
The following types of information are provided in the RunFile log for each
oligonucleotide produced:
♦
Column position, sequence name and listing for the sequence in each loaded
position
♦
Sample Collector position for labeling the samples
♦
Begin and End procedure names
♦
Two measures of the quantity of each oligonucleotide; ODUs (Optical Density
Units) and concentration in picomole/µL
♦
Name of the Synthesis cycle used
♦
Name of the Cleavage cycle used
♦
Name of the Purification cycle used
♦
Date of completion of Synthesis order (date Synthesis order was generated)
Note
You can open a RunFile in two ways, by using Simple Text or by using the control
application. When opened in Simple Text by double-clicking the File icon (Simple Text must be
present on the Macintosh), the RunFile looks like the example in the figure below. Opening the
RunFile with the ABI 3948 System Control application allows you to print labels.
4-32 Overview of Software Commands
Sample Labeling Feature
Procedure The ABI 3948 System Control application now has the capability of printing labels for
your sample vials. This feature uses the RunFile created by the instrument as the
source of the information needed for labeling.
Note
If you can’t find a Run file after a run, use the “Generate Run File” command
(page 4-16) to generate a new Run file.
To print labels for a run:
Step
1
Action
Open the Run file generated by the sample run using the Open command.
A Oligo Labels dialog box like that shown below appears. For an explanation of the
information in this view and other views shown in this procedure, see “Label View
Information” on page 4-36.
2
To select a different dilution volume, click the Dilution Volumes button to present the
Dilute Volumes view:
Note
The dialog box shown in the figures in steps 1 and 2 has two views, the
Oligo Labels view shown above, and the Dilute Volumes view shown below.
Overview of Software Commands 4-33
To print labels for a run: (continued)
Step
3
Action
Scroll the Final Concentration scroll bar (located in the top middle of the Dilute
Volumes view) to select the concentration.
You can choose any value in the range of 0.0 to 100 pmol/pmol/µL in 0.5 pmol
steps. As you scroll, notice that the amount of reagent needed to dilute each
sample changes in the sample listing below.
4
Once the dilution volume is selected, return to the Oligo Labels view by clicking the
Oligo Labels button and then scroll to the bottom of the window.
After scrolling, the Oligo Labels view looks like the figure below. This portion of the
Oligo Labels view is the Label print area which lists the information to be printed on
labels.
5
To see the details of any label in the Label print area, select it to present the
contents as shown in the figure above.
Note
The contents of the label selected to the upper left are shown in an
expanded form at the top of the window. See “Information in Individual Labels” on
page 4-36 for a description of label contents.
6
Click the Label Preferences button to present the dialog box shown below.
Note
See “Label Preferences Button” on page 4-37 for more information about
the contents of this dialog box.
4-34 Overview of Software Commands
To print labels for a run: (continued)
Step
7
8
9
Action
If desired, change the information displayed on each label as follows:
♦
Change the type of information displayed on the fourth line by clicking the radio
button for a new type of information.
♦
Change the Misc Display Options to enable or disable the display of
concentration information:
–
Checking the Show pmol values checkbox enables the display of this type
of information.
–
Checking the “Show odu values” checkbox enables the display of this type
of information.
Print out a single sheet of labels at the default X- and Y-axis settings to determine if
label printing is correctly positioned.
If the label information...
Then...
is clearly centered and visible
you are done.
is too far to the left
use the upper Printer offset scroll bar to
enter a positive X-axis value.
is too far to the right
use the upper Printer offset scroll bar to
enter a negative X-axis value.
is too far up
use the lower Printer offset scroll bar to
enter a negative Y-axis value.
is too far down
use the lower Printer offset scroll bar to
enter a positive Y-axis value.
is not oriented properly
check the orientation using the Page Setup
command (File menu) and reset it to
“Portrait” if necessary.
is too small or too large
check the Scale value with the Page Setup
command and set it to exactly 100%.
Print another sheet of labels to determine if labels print properly:
♦
If label information is clearly centered and visible, you are done.
♦
If label information does not print properly, make another setting in step 8 and
print out another sheet of labels to check printing.
Note
An alternate way of opening the Oligo Labels dialog box is to drop a RunFile icon onto
the icon for the application. This opens the controller application and inputs the selected
RunFile.
Overview of Software Commands 4-35
Label View Information
Introduction This section provides the following information on the label print views used in the
procedure above:
♦
Information in the Five Columns
♦
Information in the Individual Labels
♦
Label Preferences Button
.
Information in the The information shown in the five columns of the listing in the Labeling views in steps
Five Columns 1 and 2 above is as follows:
♦
Column 1 - Sample number
♦
Column 2 - rack position
♦
Column 3 - Sample name
♦
Column 4 - Crude/Purified (when field is blank)
♦
Column 5 - Dilution volume in µL
Information in Notice that the information contained in a selected label is presented at the top of the
Individual Labels window. To see the contents of any label in the body of the window, select the label in
the portion of the Oligo Labels view shown in step 4.
Note
The example in the procedure shows the label contents corresponding to rack position
A1 at the top of the window.
These types of information are shown in each label:
♦
Chemistry protocol name on the top line.
♦
Rack position (A1, etc., at the start of line two.
♦
Concentration in pM/µL (picomole/microliter) at the end of line two.
♦
Date of run at start of line three (011496 = November 4, 1996).
♦
Concentration in ODUs at the end of line three.
♦
Line 4 is blank until entry is made using the Label Preference button.
4-36 Overview of Software Commands
Label Preferences The Label Preferences button presents the Label Preferences dialog box, shown in
Button step 6, which enables you to do the following:
Task
Description
Centering label
information
Adjust the x- and y-axis offsets for the printer so that label
information can be centered on the label.
Specifying contents
of fourth line
Specify the content of the fourth line of the label and adjust the
position of printing so that label annotation is correctly centered on
the label
Specifying type of
information on
fourth line
Specify the type of Information presented on the fourth line of each
label, which includes:
Specifying additional
information
♦
Customer name
♦
PO (Purchase Order) number
♦
Account number
♦
A brief comment (it must fit on the fourth line)
Specify that pM/µL and ODU information be presented on labels by
checking the appropriate checkbox.
Overview of Software Commands 4-37
About Synthesis Orders
Introduction The Synthesis Order is the document used to input sequences into the ABI™ 3948
Synthesis and Purification system. A Synthesis Order documents other details about
the run used to produce it such as:
♦
Customer name
♦
Address
♦
Phone or Fax number
♦
Order date
♦
PO number
There are three Synthesis Order formats:
♦
The single order file format
♦
The short multiple order file format
♦
The long multiple order file format
The single order file is the type of Synthesis Order created in the Control application
by the New Synthesis Order command from the File menu. The two multiple order
formats are alternative ways to generate single order files. Text files using the short or
long multiple order file formats are called Multi-order files.
Ways of Creating The most direct way of creating a Synthesis Order is to use the New Synthesis Order
Synthesis Orders command from the File menu of the ABI 3948 System Control application. This
creates orders one at a time with a separate Synthesis Order required for each
oligonucleotide synthesized.
An example of a Synthesis Order is shown below. The Synthesis Order is described in
detail under “Types of Information” on page 4-5.
The Multi-order file capability was added to the ABI 3948 instrument application to
expedite creation of multiple Synthesis Orders as follows:
♦
Creating Multi-order files is a two-step process which requires you to create a text
file in the proper format, either the short or long format, and then open the files in
the ABI 3948 instrument application.
4-38 Overview of Software Commands
♦
After the ABI 3948 System application opens a Multi-order file, the application is
used to generate “single” Synthesis Order files for input to the instrument. An
example of an opened Multi-order long format file is shown below
.
Note
For a detailed description of Multi-order file formats and how to use the Multiple Order
window, see Appendix C of the ABI™ System 3948 User’s Manual, “Creating and Using
Multi-Order Files.” Synthesis Orders, however created, appear like the example shown in the
figure on “Ways of Creating Synthesis Orders” on page 4-38.
Once single format Synthesis Orders are created, they can be sent to the Synthesis
database during run setup and then downloaded to the instrument when the run
starts. For more information on this, see the instructions provided in Chapter 2 of the
ABI™ 3948 System User’s Manual (“Assigning Sequences for the Run” on page 2-22).
Synthesis Order When you directly create a single order file using the New Synthesis Order command,
Sequence Entry a sequence can be input into the order in one of three ways:
♦
By typing it in
♦
By importing a sequence from a file using the Import Sequence command, or
♦
By using cut and paste to transfer the sequence from other applications
Once you have input a sequence, you can use it as is or you can edit it.
Note
One type of edit is to change the A, G, C, and T letter representation to bottle position
representation (5, 6, 7, 8).
Besides entry of the sequence in terms of the four bases, sequences can be entered
or imported using mixed bases as a designation for a base site or ambiguity
characters can be used to designate a base site.
Importing and Upon opening a new Synthesis Order, the Import Sequence command (File menu) is
Exporting available to import sequences. This command, however, can only be used to import
Sequences sequences previously exported to a file to be archived from the ABI 3948 System
Control application.
Once you have completed a Synthesis Order, the Export Sequence command is
available to write a plain text file containing only the sequence for use as input to all
programs that accept plain text input.
IMPORTANT
The Export Sequence command is probably not the best way of maintaining a
library of sequences since it provides just the sequence text.
Overview of Software Commands 4-39
Rather than exporting a sequence, it is better to use a Synthesis Order file to store
each sequence since you are provided with fields for:
♦
Customer information
–
Name
–
Address
–
Phone and Fax number
♦
Purchase Order and Account information
♦
Order Date
♦
Comments
♦
Protocol Option
♦
Sequence name
♦
Numbers of each of the bases or bottle positions 5 through 8
Synthesis Orders can be opened as well in many other applications.
Actions after Once you have created or edited a sequence, you can:
Sequence Entry ♦ Highlight the sequence listing and read it back using the Read Selection
command (Edit menu) to ensure that it was entered correctly.
♦
Save the Synthesis Order containing the sequence for use in producing an
oligonucleotide in the instrument
♦
Print out a copy, or
♦
Export the sequence to a file to be archived
4-40 Overview of Software Commands
Multi-Order Files
Introduction Multi-order (multiple Synthesis Order) files are discussed in this chapter because they
may be opened by the Open command. Only general information is provided here. For
a detailed description of the use of Multi-order files, see Appendix C of the ABI™ 3948
User’s Manual, “Creating and Using Multi-Order Files.?
Multi-order text files are created in either of two formats by a text processor and have
the following characteristics:
Characteristic
Description
Correct syntax
and starting tag
Multi-order files must have the correct syntax and start with a
SYNTHMOSFORMAT tag for the short format or a SYNTHMOLFORMAT
tag for the long format.
Limitation of
short format
The short format only provides the capability to name the resulting
oligonucleotide and list the sequence.
Additional long
format
capabilities
The long format additionally provides the capability to:
Maximum
number of
Synthesis
Orders
♦
List the names of the customer and synthesizer.
♦
Add comments.
♦
Set the DMT and PUR options.
The maximum number of Synthesis Orders which can be created with a
single Multiple Order file is 999.
Types of All Multiple Order files may be generated any of the following types of applications:
Applications Used to ♦ A text editor or a word processor that has its formatting capabilities disabled
Create
♦
A stand-alone application such as a Web page order entry program
♦
A spreadsheet or database management program, especially one with
programming or scripting capabilities
Multiple Synthesis Order files appear as shown below when opened with the ABI 3948
System Control application Open command:
Overview of Software Commands 4-41
Note
Both Long and Short Format files appear as shown in the figure but the Long Format
file will create Synthesis Orders with more content when additional entries are made in the text
file.
Generating Once a Multiple Order file is created, in either format, all that is required to generate
Synthesis Orders single order Synthesis files is to open the file from the ABI 3948 System Controller
application and then click the Make Order Files button (see figure on previous page).
4-42 Overview of Software Commands
Communication View and
Operational Views
5
5
In This Chapter
Topics Covered This section provides detailed information on a number of Synthesizer views used to
operate the instrument from the ABI™ 3948 System Control application. The pop-up
menu in all views is used to access any other Synthesizer view.
This chapter contains two sections:
Topic
See page
General Information about Operational Synthesizer Views
5-2
Using the Run SetUp View
5-20
Communication View and Operational Views 5-1
Section 1 - General Information about Operational Synthesizer Views
Contents of Section 1 Section 1 provides referrence information on the operational Syntheisizer Views and
covers the following topics:
Topic
About the Synthesizer Window
See page
5-4
Introduction
5-4
Access to Views
5-4
Type of Information in This Chapter
5-5
Keyboard Shortcuts
5-5
Communication View
Initial Database Window
Accessing the Synthesizer Window by Password
Run Setup View
5-6
5-6
5-6
5-7
Tasks Performed
5-7
Accessing the Run Setup View
5-7
Menus and Buttons
Run Protocol View
5-8
5-9
Used to Create Protocols
5-9
Protocol Name
5-9
Synthesis Cycle
5-9
Cleavage Cycle
5-9
Purification Cycle
5-9
Monitor Chemistry View
5-9
Introduction
5-9
Three Chemistry Panes
5-12
Description of Sequence Pane Information
5-13
Status and System Messages
5-13
Using Monitor Chemistry Information
Monitor Instrument
5-14
5-15
Introduction
5-15
Current Date and Time
5-15
Valves
5-15
Liquid Sensors
5-16
Conventions
5-16
Synthesis Jaw/Valve Block/Manifold Sensors
5-16
Cleavage Jaw Sensors
5-17
Deprotection Coil Sensors
5-17
Purification Jaw Sensors
5-17
Phosphoramidite Sensors
5-17
UV Detector Sensor
5-17
Pressure Regulators
5-17
5-2 Communication View and Operational Views
Topic
See page
Other Parameters and Instrument Conditions
Monitor Run View
5-18
5-19
Introduction
5-19
Types of Information Provided
5-19
Communication View and Operational Views 5-3
About the Synthesizer Window
Introduction The Synthesizer window of the ABI 3948 System Control application:
♦
Appears on the computer monitor whenever you establish communication
between the computer and a ABI 3948 System.
Note
♦
Up to four instruments at a time may be used with a single Macintosh.
Contains all the tools you need to control and monitor the ABI™ 3948 Nucleic Acid
Synthesis and Purification System.
The initial Synthesizer window, shown below, is the Communication view. This view
appears whenever the ABI 3948 System Control application accesses an instrument.
Pop-up menu
2.20
x.xx
Access to Views A pop-up menu at the top of the Synthesizer window displays the following views:
List of Synthesizer Window views:
View
Purpose
Communication
Initial view listing software version
Run Setup
Used to set up a run
Run Protocol
Used to create new protocols
Monitor Chemistry
Used to monitor chemistry during a run
Monitor Instrument
Used to monitor instrument hardware during a run
Monitor Run
Used to monitor the Synthesis Orders produced during a run
Edit Synthesis Cycle
Used to create and edit Synthesis cycles
Edit Cleavage Cycle
Used to create and edit Cleavage and Deprotection cycles
Edit Purification Cycle
Used to create and edit Purification cycles
Edit Begin Procedure
Used to create and edit Begin procedures
Edit End Procedure
Used to create and edit End Procedures
Edit Bottle Procedure
Used to create and edit Bottle Procedures
Misc (miscellaneous)
Procedure
Used to create and edit other types of procedures
Manual Control
Used to manually execute functions
5-4 Communication View and Operational Views
List of Synthesizer Window views: (continued)
View
Purpose
Power Fail History
Provides a listing of instrument power failures
Instrument Test
Used to test instrument hardware
B+Tet Calibration Table
Lists maximum times for B+Tet deliveries
Reagent Utilization Table
Lists volumes for various reagent deliveries
Type of Information To avoid presenting information in the same way in both the User and Reference
in This Chapter manuals, Chapter 2 of the ABI™ User manual provides procedural information for the
Synthesizer window and this chapter of the ABI™ Reference manual may provide
procedures but primarily provides user interface details.
Keyboard Shortcuts As an alternative to the pop-up menu in the Synthesizer Window, you can
simultaneously press the Command key () and one other key on the computer
keyboard to see nine of the views.
Synthesizer Window View
Keyboard Shortcut
Run Setup
c 1
Run Protocol
c 2
Monitor Chemistry
c 3
Monitor Instrument
c 4
Edit Synthesis Cycle
c 5
Edit Cleavage Cycle
c 6
Edit Purification Cycle
c 7
Edit Bottle Procedure
c 8
Manual Control
c 9
Communication View and Operational Views 5-5
Communication View
Initial Database The initial instrument database window which appears is the Communication view.
Window
2.20
x.xx
The Communication view of the Synthesizer window appears when you choose an
instrument and a password dialog box is presented as the window appears.
Two types of passwords provide access to the Synthesizer window:
Accessing the
Synthesizer Window
by Password
♦
Full Access - provides access to all views of the window
♦
Monitor Access - provides access only to the Monitor Chemistry, Monitor
Instrument, Monitor Run, and Power Fail History Views.
Open up a Synthesizer window as follows:
Step
Action
1
Choose the Open Synthesizer command and select the instrument to be opened
from the dialog box. This will present the Communication view and a Password
dialog box like that shown to the left below:
2
Type in the password in the dialog box and click OK to open access to the
Synthesizer database:
♦
If no password has yet been assigned, click OK without entering a password.
♦
If an incorrect password is entered, the dialog box shown to the right below
replaces the first dialog box. Clicking OK in this dialog box presents the dialog
b ox to the left again.
Note
5-6 Communication View and Operational Views
You must enter the correct password to get past this point.
Run Setup View
Tasks Performed This subsection provides general interface information on the first page on the Run
Setup View. For information on other Run Setup view windows, see Section 2 of this
chapter.
The Run Setup view, shown below, enables you to perform the following tasks:
Task
Description
Select sequences
for the next run
Select/remove sequences to be used for the next run.
Assign chemistry
and sort sequences
Assign chemistry protocols and sort the sequences by protocol to
determine turntable loading positions.
Determine if
sufficient reagents
are on instrument
Determine if additional reagents are needed for the next run.
When insufficient reagents are available for the entire run, the
options are:
♦
To replace reagents before the run
♦
To program a Pause After to replace reagents
Install OneStep
columns
Place OneStep columns in the proper positions on the turntable to
prepare for the run.
Prepare for run
extension
Program a “Pause After” during a run to occur after synthesis has
completed on a designated turntable row in preparation for run
extension
Perform setup
needed to extend a
run
Enter sequences and load columns needed for run extension during
a programmed system pause and then restart the run.
Accessing the Run The Run Setup view is chosen either by executing this command from the
Setup View Communications view menu or by the Command - 1 key sequence.
Communication View and Operational Views 5-7
Menus and Buttons Various details of the Run Setup view are described in the following table:
Run Setup View Pop-Up Menus and Buttons
Pop-up Menus and
Buttons
Descriptions
Open
This button is used to input Synthesis Orders into the Sequence
Order list.
Del From List
After you have moved sequences into the Sequence Order list
using the Open button, the Del From List button is used to remove
any selected sequence from this list.
Add+
Click the Add + button to transfer one or more selected sequences
from the Sequence Order List to the Run Setup list.
Remove
Click the Remove button to transfer a selected sequence from the
Run Setup Order box back to the Sequence Order List.
Protocol Pop-up menu
This pop-up menu allows a protocol to be assigned to a selected
row in the Run Setup list.
AutoSort
This button is used to autosort the sequences entered into the
Run Setup list to maximize throughput on the instrument. See
"AutoSort Procedure" on page 2-24 of the ABI™ 3948 System
User’s manual for more information
Begin and End
Procedure Pop-up
Menus
These menus allow a different Begin and End procedure to be
assigned in this view, but only if you have previously developed
them.
Bottle Usage
This button presents the Bottle Usage window to determine if
sufficient reagents are present on the instrument to accommodate
the current run. See "Checking Reagents and Required OneStep
Columns" on page 2-27 of the ABI™ 3948 System User’s manual
for more information.
The Bottle Usage window also indicates how many of each type of
OneStep column is required.
Load
This button presents the Load window enabling OneStep columns
to be properly placed on the instrument. See "Loading" on
page 2-30 of the ABI™ 3948 System User’s manual for more
information.
Extend Run
This button is used to extend a run when the run has been
interrupted by a programmed “Pause After.” See "Using “Pause
After” During a Run" on page 2-35 of the ABI™ 3948 System
User’s manual for more information.
5-8 Communication View and Operational Views
Run Protocol View
Used to Create The Run Protocol view is used to create protocols from the available cycles and
Protocols procedures. This view initially looks like the figure below:
Each protocol comprises: a Synthesis Cycle, a Cleavage Cycle, and a Purification
Cycle. Use the Run Protocol view as follows:
♦
When you select a protocol from the Protocol Name pop-up menu, its component
cycles and procedures appear.
♦
If you select an undefined protocol, its component cycles and procedures become
the default values listed for the standard protocol.
♦
To create a new protocol, enter a new name for the protocol, then choose the
cycles and procedures from each pop-up menu, and click the Change button.
Note
The Edit Synthesis Cycle, Edit Cleavage Cycle, and Edit Purification Cycle views are
available to develop new cycle chemistry.
Protocol Name This pop-up menu lists the standard protocols (standard, dye-labeled, and biotin) and
the additional undefined protocols.
Synthesis Cycle This pop-up menu lists standard Synthesis Cycles and additional undefined cycles.
Use the Edit Synthesis Cycle view to define a new Synthesis Cycle or edit an existing
one (see page 6-17).
Cleavage Cycle This pop-up menu lists standard cycles and additional undefined cycles. Use the Edit
Cleavage Cycle view to define a new Cleavage Cycle or edit an existing one (see
page 6-18).
Communication View and Operational Views 5-9
Purification Cycle This pop-up menu lists standard cycles and additional undefined cycles. Use the Edit
Purification Cycle view to define a new Purification Cycle or edit an existing one (see
page 6-19).
5-10 Communication View and Operational Views
Monitor Chemistry View
Introduction The Monitor Chemistry view, shown below, has the following four main areas:
Area
Description
Upper right
corner
A system status indicator in the upper right corner.
Three Chemistry
panes
Three Chemistry panes (Synthesis, Clv/Dep, Dep/Pur) occupying about
one half of the window which present information about the chemistries
in progress.
Small middle
pane
A small middle pane which provides information about the sequences
currently undergoing synthesis on the Synthesis pane.
Lower pane
A lower pane which presents a series of messages from the instrument.
Note
This is the only view that will not display a “Message Waiting” or “Critical Message
Waiting” icon to the upper left because all messages are presented at the bottom of this view.
Syn v4.20f
Cleave v4.20g
C
, DB 4.20, 2.20.
, DB 4.20, 2.20.
The following conventions apply to interpreting the Monitor Chemistry View:
Convention
Description
Grouping of three
As shown in the figure above, the turntable position numbers are
placed in the three panes in groups of three
Each group is a row
Each group of three represents a radial row of column positions on
the turntable which undergo the same type of chemistry.
Same cycle on each
group
The instrument uses the same cycle on each group of three
oligonucleotides and concurrently uses three different cycles at the
processing stations.
Communication View and Operational Views 5-11
The general status of the instrument is indicated in all views by presentation of one of
the following four words in the upper right corner:
♦
Ready
♦
Running
♦
Manual Control
♦
Interrupted.
Three Chemistry The three chemistry panes (Synthesis, Clv/Dep, Dep/Pur) show details of the
Panes chemistries in progress in the three chemistry modules. After the first set of three
columns reaches the Purification module, three chemistries are concurrently in
progress on the instrument (until the end of the run).
Progress of Chemistry
Chemistry progresses as follows when a run is started:
Stage
Action
1
First the Begin procedure can be seen executing in the left pane.
2
Next, when the first three columns are undergoing synthesis, only this type of
chemistry is in progress and information is presented only in the left pane.
3
When the first three columns move to the Cleavage module, the next set of three
oligonucleotides starts synthesis. Synthesis and Cleavage chemistries are
concurrently in progress at this point.
4
Finally, when the first set of three columns reaches the Purification module, all three
chemistries are concurrently in progress with all three panes showing chemistry
details.
Description of Chemistry Panes
All three panes show the same types of information, described in the two tables below:
Location
Purpose
Upper Part
of Pane
This portion of each pane shows two types of information:
♦
The turntable position of each oligonucleotide, expressed as a number
between 1 and 48, and
♦
The name of each sequence (Synthesis Order name; the name of the
sequence may be different than the Synthesis Order name).
Note
If no name is specified when making a Synthesis Order, the default
name is the sequence name when one is entered.
5-12 Communication View and Operational Views
Location
Name
Description
Lower Part
of Pane
Cycle Name
Name of cycle in progress.
Step
Number of cycle step currently in progress/total number of
steps in the procedure.
SubStep/
Loop
The standard chemistry cycles provided with the instrument
use subroutines in executing chemistry.
SubSteps are steps in subroutines and, optionally, if present
the Loop number specifies the current iteration of the current
loop.
Fxn
The name of the function currently being executed.
Fxn Time
The remaining time that the function is active.
Listed for example as 15/20 where 20 is the total time the
function is executed and the first number is a decrementing
counter, which counts down from the second value to “0”
upon completion of execution.
Fxn Number
The number of the function. More information about
functions is presented in this section (see “Manual Control
View” on page 6-3) and Appendices A and C.
Description of The middle pane provides a listing for each of the three sequences currently being
Sequence Pane synthesized in the Synthesis module:
Information ♦ The sequence on each row is identified by number as to the location of the column
in the turntable.
♦
Each sequence is then listed with an orientation in the 5´ to 3´ direction.
♦
The numbers listed under the Base heading at the end of each row indicate the
current base position being synthesized.
The number 2/25, for example, indicates that the second base position of a total of
25 bases in the sequence is currently being synthesized.
Note
Following convention, a DNA sequence is entered 5´ to 3´. Likewise, the sequence
listing in the ABI™ 3948 System displays each sequence in a 5´ to 3´ orientation. However, the
DNA sequence is actually synthesized 3' to 5'. This is indicated in the Monitor Chemistry View
by the highlighting of the current base position being synthesized.
Status and System The lower pane presents a series of messages from the instrument listing the times at
Messages which major processing events occurred. This listing may also include error and other
system messages. The following messages are examples of system status messages.
Each message is prefaced by the time at which an action was taken:
11:31:35 AM Starting Manual Control Action
11:31:38 AM Manual Control Action Complete
11:32:17 AM Starting Chemistry Run
12:41:55 PM Interrupting system
12:45:05 PM Resuming Chemistry
12:50:10 PM Chemistry Done
Communication View and Operational Views 5-13
The log on the bottom of this view informs you of the following types of events:
♦
When an instrument run was interrupted or aborted
♦
When a sensor fails to detect liquid (which may or may not interrupt the run)
♦
Instructions used during procedures (such as autodilution)
♦
Power fail information
♦
Start and stop times of chemistry runs
♦
Execution of runs or manual control events
The information presented on this log is intended to inform a user about the normal
progress of a run and for troubleshooting. For a full list of messages presented in the
log, see "System Messages" on page C-38 of Appendix C
Using Monitor While information in the Monitor Chemistry view is primarily used to keep track of
Chemistry chemistry in progress, the log on the bottom also informs you when an interrupt or
Information Pause After occurs so that you can perform a bottle change procedure, remove a high
priority oligonucleotide, or extend the run.
Instructions to the user during procedures (calibration, bottle change, etc.) are
presented in the log. You can also use the log for troubleshooting the instrument in
conjunction with the Monitor Instrument view.
5-14 Communication View and Operational Views
Monitor Instrument
Introduction The Monitor Instrument view, shown with a run in progress in the figure below,
displays the status of system valves, liquid sensors, pressure regulators, as well as
other parameters and instrument conditions. These parameters and conditions include
♦
Chemistry status
♦
Deprotection coil temperature
♦
Current turntable and sample collector positions
♦
Status of Synthesizer, Cleavage, and Purification jaws, and
♦
Status of heater and fan
This view is best used in conjunction with the Monitor Chemistry view to troubleshoot
problems which may occur at any point during a particular chemistry.
Current Date and The current ABI™ 3948 System date and time is displayed at the top of the window, to
Time enable you to correlate particular events occurring in one of the chemistry cycles with
the underlying hardware and/or software supporting chemistry.
Valves The Monitor Instrument view supports up to 80 numbered valves in its current form.
The status of valves is indicated in groups of five symbols, 10 valves per line, with the
status of a particular valve marked with either a bullet or an exclamation point:
♦
Valves that are off are indicated by a bullet, and
♦
Valves that are on are indicated by an exclamation point
Separating each line of 10 valves into two groups of five makes the display easier to
read.
Communication View and Operational Views 5-15
Liquid Sensors
Conventions The instrument has a number of liquid sensors placed in critical portions of its
plumbing to ensure that liquid transfers are being made. See "Edit Cycle and
Procedure Interface" on page 6-12 for information on enabling a sensor for a particular
function.
When enabled in the cycle or procedure, a check in a liquid sensor check box
indicates that liquid is detected at the current time at the sensor. In the discussion in
this section, sensors are grouped according to:
♦
The position in the plumbing, or
♦
The type of phosphoramidite monitored
Except for the check boxes labeled A, G, C, and T, which indicate phosphoramidites,
all of the letters A,B, and C included as part of a sensor designation indicate one of
the three positions in a jaw mechanism:
Position
Description
Position A
Position A corresponds to the outer in a row of columns in the turntable.
Position B
Position B is the middle position.
Position C
Position C is the inner position.
You can locate the sensors by number on the instrument plumbing diagram provided
in Appendix D. A checked box for a particular sensor means that liquid is present at
the sensor at the current time. An unchecked box means that gas is present at the
sensor at the current time.
Synthesis Jaw/Valve The synthesis sensors listed in the table below are in the plumbing on either the upper
Block/Manifold or lower side of the synthesis jaw mechanism:
Sensors ♦ The first three sensors are located on the upper side of the jaw mechanism.
♦
The next three sensors, labeled “Man.”, are located just below the first manifold
under the synthesis jaw.
♦
The last three sensors, labeled “Syn. Lower,” are located closest to the reagent
delivery valve blocks.
Name
Identity
Purpose
SUA
Syn. Upper A - Sen. 3
SUB
Syn. Upper B - Sen. 2
Determines that reagent for the listed position has
reached the sensor position.
SUC
Syn. Upper C - Sen. 1
ManA
Man. A - Sen. 22
ManB
Man. B - Sen. 23
ManC
Man. C - Sen. 24
SLA
Syn. Lower A - Sen. 6
SLB
Syn. Lower B - Sen. 5
SLC
Syn. Lower C - Sen. 4
5-16 Communication View and Operational Views
Determines that reagent for the listed position
reaches the sensor position.
Determines that reagent for the listed position
reaches the sensor position.
Cleavage Jaw The three sensors listed in the table below are in the plumbing on the upper side of the
Sensors cleavage jaw mechanism:
Name
Identity
Purpose
CA
Cleavage A - Sen. 7
CB
Cleavage B - Sen. 8
Determines that reagent for the listed position has
reached the sensor position.
CC
Cleavage C - Sen. 9
Deprotection Coil The three sensors listed in the table below are in the plumbing on the exit side of the
Sensors deprotection coils:
Name
Identity
Purpose
DEPA
Coil A - Sen. 10
DEPB
Coil B - Sen. 11
Determines that reagent for the listed position has
reached the sensor position.
DEPC
Coil C - Sen. 12
Purification Jaw The three sensors listed below are in the plumbing on the upper side of the
Sensors purification jaw mechanism:
Name
Identity
Purpose
PA
Pur. A - Sen. 14
PB
Pur. B - Sen. 15
Determines that reagent for the listed position has
reached the sensor position.
PC
Pur. C - Sen. 16
Phosphoramidite The four sensors listed below are in the plumbing between the phosphoramidite
Sensors bottles and Valve Block B:
Name
Identity
Purpose
A
Phos. A - Sen. 17
Determines that reagent for the listed position has
reached the sensor position.
G
Phos. G - Sen. 18
C
Phos. C - Sen. 19
T
Phos. T - Sen. 20
UV Detector Sensor A single sensor, labeled UVQ, is in the plumbing input to the UV detector.
Pressure Regulators The current values in psi (pounds per square inch) for the ten pressure regulators are
shown in this portion of the Monitor Instrument view:
♦
The ten pressure regulators initially have the values listed in the table.
♦
Except for Regulators 1, 3 and 10, the regulators are maintained within ±0.2 psi of
the initial pressures.
Communication View and Operational Views 5-17
♦
Regulators 1, 3 and 10 are programmed to vary pressures during the Cleavage
and Purification cycles.
Reg. No.
Pressure
Reg. No.
Pressure
1
10
2
8
3
10
4
7
5
8
6
6
7
10
8
9
9
6
10
10
Other Parameters The parameters and conditions listed under the Other category on the Monitor
and Instrument Instrument view are as follows:
Conditions ♦ Deprotection coil temperature
♦
Opcodes
♦
Current turntable and sample collector positions
♦
Status of Synthesizer, Cleavage, and Purification jaws, and
♦
Status of heater and fan
Information for the parameters above is presented in the table below:
Parameter
Description
Temperature (Temp)
The value presented for this parameter is the current temperature
of the deprotection coils.
During the Cleavage and Purification cycles, the temperature of
the coils is programmed using the Set Coil Temp parameter
(Function 195), designating the new temperature with the MISC
data entry for the function.
Opcodes
Messages presented in this field are only of interest to system
developers. No user level information is presented here.
TT Position
This is the current synthesis module turntable position (1-18)
SC Position
This is the current sample collector position (1-48)
SC Rack Type
Indicates the type of rack in the sample collector, either Red 6x8
(standard OligoRack) or White 4x12 (screw top microtiter).
Syn Jaw
This is the current status of the Synthesizer jaw mechanism, either
Open or Closed.
Clv Jaw
This is the current status of the Cleavage jaw mechanism, either
Open or Closed.
Pur Jaw
This is the current status of the Purification jaw mechanism, either
Open or Closed.
Heater and FAN check
boxes
If either of these check boxes is checked, the device associated
with the check box is turned on.
5-18 Communication View and Operational Views
Monitor Run View
Introduction The Monitor Run view, shown in the figure below, is another way of observing the
progress of an ABI™ 3948 System run. Information in this view is useful in tracking the
general course of chemistry, knowing when to set a Pause After in the run, and is a
table of the entire run which provides useful information in tracking the run.
Types of Information The Monitor Run view provides the following types of information about the run in the
Provided form of a table:
Type of Information
Description
Row
Each of the 16 rows in the turntable is labeled by number in the
first column.
Column A
Names of the three Synthesis Orders on each row of the turntable
are listed row by row in second through fourth columns of the table.
Column B
Column C
Chemistry Status
Pause After
The fifth column lists the current chemistry status for each row – a
particular row has one of these notations:
♦
1) Scheduled/Not scheduled
♦
2) Synthesizing
♦
3) Cleaving
♦
4) Purifying
♦
5) Completed
The sixth column, Pause After, has either “No”, “Yes”, or nothing
after a row to indicate whether a pause has been programmed.
Communication View and Operational Views 5-19
Section 2 - Using the Run Setup View and Extending a Run
Contents of Section 2 This section describes how to use the Run Setup view and extend a run.
The section covers the following topics:
Topic
Importing Synthesis Orders
See page
5-21
Process of Importing Synthesis Orders
5-21
Description of Open Dialog Box Buttons
5-22
Selecting Orders for the Next Run
Description
Synthesis Order Information Provided for a Selected Sequence
Provided for a Selected Sequence
Assigning Chemistry and AutoSorting
5-23
5-23
5-24
5-24
5-25
Introduction
5-25
Assigning Chemistry
5-25
AutoSorting
5-25
Bottle Usage
Procedure
Using the Load Display
5-27
5-27
5-28
Types of Information Provided
5-28
Procedure for the Load Display
5-28
Significance of the Three Positions
5-28
Protocol and Cycle Identification
5-29
Using the Buttons on the Display
5-29
Extending a Run
Using Pause After
5-20 Communication View and Operational Views
5-30
5-30
Importing Synthesis Orders
Process of Importing This subsection lists the process of importing Synthesis Orders into the Run Setup
Synthesis Orders view and provides detailed information about the Open dialog box used for importing.
The Open button on the Run Setup view is initially the only button active in the
Synthesizer window. When it is clicked, the dialog box below is presented to enable
entry of Synthesis Orders into the Run setup view:
Figure 10-1 Import Synthesis Order Dialog Box
The dialog box in Figure 10-1 is used as follows:
Stage
Description
1
Synthesis Orders are accessed in the usual way in the top scroll box.
2
Once orders are present in the top scroll box, the orders to used in the next run are
moved to the bottom scroll box by clicking the Add or Add All buttons.
The Add button moves only those orders selected while the Add All moves all
orders from the top to the bottom scroll box.
3
Any orders placed in the bottom scroll box by mistake can be removed using the
Remove button.
Communication View and Operational Views 5-21
The dialog box in Figure 10-1 is used as follows: (continued)
Stage
4
Description
When the set of orders to be input to the Run Setup view is present in the
lower scroll box, click the Done button to move them to the Sequence
Order list, the top scroll box in the Run Setup view. as shown below:
For the procedure on using the dialog box as outlined above, see "Assigning to the
Run Setup View" on page 2-20 of the ABI™ 3948 System User’s manual.
Description of Open The Open dialog box is used as described in the table below to load sequences into
Dialog Box Buttons the Sequence Order list (see the lower figure on page 5-9).
Dialog Box Button or
Checkbox
Description
Add/Add All
One of these buttons is used to move Synthesis Orders from the
upper scroll box to the lower scroll box in the dialog box. The Add
button moves selected Synthesis Orders and the Add All button
moves all orders to the lower scroll box when a single order is
selected.
Show all Files
Checkbox
Click the check box labeled Show all files to view all file names,
regardless of the file’s format.
Remove
If you have inadvertently moved one or more Synthesis Orders to
the lower scroll box, this button moves selected orders back to the
upper box.
Eject
If you are adding Synthesis Orders from a diskette in the floppy
drive, this button enables you to eject a floppy so that you can
insert another.
Done
When you have finished moving Synthesis Orders to the lower
scroll box, click Done to close the dialog box and transfer all orders
in the lower scroll box to the list of Ordered Sequences in the Run
Setup view.
Cancel
This button enables you to return to the Run Setup window without
adding sequences.
5-22 Communication View and Operational Views
Selecting Orders for the Next Run
Description The next step in inputting Synthesis Orders for a run is to move the orders to be
produced by the next run from the Sequence Order List (at the top of the Run Setup
view) to the Run Setup list at the bottom.
This is done by selecting the orders in the top list and then clicking the Add+ button to
move them to the lower list. For the procedure on this, see "Assigning Sequences for
the Run" on page 2-22 of the ABI™ 3948 System User’s manual.
Communication View and Operational Views 5-23
Synthesis Order Information
Provided for a As shown in the figure below, whenever a single sequence is selected in the Run
Selected Sequence Setup scroll box, several types of information are presented for that sequence below
the scroll box. This information includes:
♦
A detailed base by base listing between the 5´ and 3´ ends
♦
The Specification that oligonucleotide be
♦
–
Purified, or
–
Crude
The length of the sequence
Note
If you decide not to produce a particular sequence during the upcoming run, click
Remove to return it to the upper list.
5-24 Communication View and Operational Views
Assigning Chemistry and
AutoSorting
Introduction Once the desired sequences have been moved to the Run Setup scroll box, the next
thing to do in preparing for a run is to assign chemistry and then autosort, if
necessary, to determine turntable loading positions.
Assigning Chemistry
The general process of chemistry needed to produce an oligonucleotide is described
in the following table:
Chemistry
Description
Protocol
Select the sequence then choose the appropriate protocol from
the Protocol pop-up menu to make an assignment.
The following rules apply to assigning protocols:
♦
All sequences in a given row must have the same protocol
assigned to them.
♦
A protocol must be assigned to each sequence in the Run
Setup scroll box.
♦
A protocol assignment can be made for Multiple orders by
using the shift key to select them and then select the protocol.
♦
To assign a single protocol to all sequences, select a single
sequence and use Command-A (-A) before choosing the
protocol.
The word Protocol appears next to a pop-up menu of protocols
that are listed in the Run Protocol view (see Run Protocol View).
You must assign a protocol to each sequence in the Run Setup
scroll box.
Begin Procedure
Use the default choice unless you have developed an additional
Begin procedure you want to use.
This pop-up menu lists the pre-defined Begin procedure as well as
possible additional undefined procedures. Use the Edit Begin
Procedure view to define a new Begin Procedure (see page 6-20).
End Procedure
Use the default choice unless you have developed an additional
End procedure you want to use. This pop-up menu lists one
standard End procedure and possible additional undefined
procedures. Use the Edit End Procedure view to define a new End
Procedure (see page 6-21).
AutoSorting Do the following to autosort sequences:
Stage
1
Description
Use the AutoSort button to sort the sequences in the Run Setup Order box.
Note
The AutoSort button does not become active until each of the sequences
in the Run Setup Order has an assigned protocol.
The top row of sequences in the Run Setup Order box is the first row of sequences
synthesized.
Communication View and Operational Views 5-25
Stage
2
Description
AutoSort applies the following sorting rules to guarantee that any set of oligo
orders, once sorted, resort to the same positions:
♦
First, sequences are arranged in rows by protocol.
♦
Second, sequences are arranged by the number of bases in each
sequence.
The longest sequence in a row is on the left; the shortest sequence in a row is
on the right.
♦
Third, sequences are arranged according to the 3´ end base, with A before C,
C before G, and G before T.
If two or more sequences in a row contain the same number of bases AND the
same 3´ base, the sequence that contains an A at the 3´ position goes before a
sequence containing a G at the 5´ end, and so on.
♦
Fourth, sequences are arranged by their sequence order file (Macintosh file)
names.
Note
3
To change the position of a single sequence for any reason, the Arrow buttons are
used as described below:
Step
Action
a.
Select a sequence by clicking it once.
b.
Click an arrow once to move the sequence one location. You can click
any arrow repeatedly or click any combination of arrows
5-26 Communication View and Operational Views
Bottle Usage
Procedure The Bottle Usage button becomes active after protocols have been assigned to the
sequences in the Run Setup Order scroll box:
Step
1
Action
Click Bottle Usage to see the display of reagent requirements for the syntheses
ordered, like that shown below:
31
The calculations for required volumes are based on the data found on the Reagent
Utilization Table (see page 6-9). Compare the volumes required for each reagent
shown in this Bottle Usage display to the actual amounts of each reagent attached
to the instrument.
2
Compare the volumes required for each reagent shown in this Bottle Usage display
to the actual amounts of each reagent attached to the instrument.
3
To return to the original Run Setup view, click OK.
Communication View and Operational Views 5-27
Using the Load Display
Types of Information This subsection provides the following information:
Provided ♦ When to Use the Load Display
♦
Significance of the Three Positions
♦
Protocol and Cycle Identification
♦
How to Use the Buttons on the Display
Procedure for the Do the following to use the load display:
Load Display
Step
Action
1
Click the Load button (figures on page 5-24) when you are ready to load OneStep
columns on to the ABI 3948 System turntable.
2
After you click this button, the Run Setup view changes to display a wedge-shaped
area on the turntable (like that shown below):
Significance of the This Run Setup view displays one row of three oligonucleotide positions on the
Three Positions turntable at a time:
♦
Each position is represented by a circle with a number.
♦
Each colored circle contains a colored letter that corresponds to the type of
OneStep column required at each position: green=A, yellow=G, red=C, and
blue=T.
♦
Each type of column provides the support required for the initial base at the 3'
position.
Use this view as a map to load columns into the turntable.
5-28 Communication View and Operational Views
Protocol and Cycle The name of the protocol and chemistry cycles assigned to the three oligonucleotides
Identification on the current turntable row are listed at the bottom of the view. This information is
useful when using the Scan function in this view (Start Scan button) to double check
your assignment of Protocol with its included set of Synthesis, Cleavage, and
Deprotection cycles.
Using the Buttons on The buttons in the Load Display are used as described in the table below:
the Display
Button
Description
Next
Click the Next button to advance to the next row of columns in the
turntable. Each click moves the turntable one row and updates the
view.
Note
The front panel “Column load” button on the instrument
has the same affect as the Next button
Prev
Click the Prev button to move the turntable in the reverse
direction. Each click moves the turntable a single row and
updates the view
Start Scan
Click the Start Scan button to quickly look at all of the positions on
the turntable and visually check that the proper column was
loaded into the position.
The scan displays all columns before stopping, pausing briefly at
each row. Stop Scan terminates the turntable scan.
Cancel
Click the Cancel button to return to the original Run Setup Order
view without starting the instrument.
Note
If you want to view the Bottle Usage display, you must
click the Cancel button to return to the Run Setup Order.
Start
Click the Start button to begin instrument operation.
After you click Start, the turntable rotates to the starting position
and the information about sequences, protocols, and column
positions is transferred from the computer to the instrument.
Note
A status indicator is presented to indicate that
information is downloading.
As soon as loading is complete, the Synthesizer window view
changes to Monitor Chemistry, and synthesis is initiated.
Refer to the manual section entitled “Monitor Chemistry View” for
information on monitoring chemistry during synthesis. If you
desire to monitor hardware conditions occurring during synthesis,
change to the Monitor Instrument view (see page 5-15). The
Monitor Run view shows the overall progress of the instrument
through the run.
Communication View and Operational Views 5-29
Extending a Run
Using Pause After The column of check boxes on the right side of the Run Setup Order scroll box,
labeled Pause after, is used to indicate when a pause is programmed using the Pause
After command (Synthesizer menu). When an X appears in a box, an instrument
pause occurs when that row of sequences has been synthesized.
Note
You can only program a Pause After once a run has started.
Pause After is used to extend a run (see "Extending a Run" on page 2-35 of the ABI™
3948 System User’s manual).
IMPORTANT
Only a Pause After, not an interrupt, may be used to extend a run.
For information on setting a Pause After, see “Pause After Command” on page 4-23.
For more information on run extension, see “Extending a Run” on page 2-35 of the
ABI 3948 User’s Manual.
5-30 Communication View and Operational Views
Synthesizer Views
Supporting Operation
6
6
In This Chapter
Topics Covered This chapter provides information on the Synthesizer Views not covered in Chapter 5,
which support the run but are not necessarily accessed during the run. The various
Edit Cycle and Procedure views used to support chemistry are discussed in Section 2
of this chapter.
This chapter contains two sections:
Section
See page
Non-Cycle Operational Support Views
6-2
Editing Cycle and Procedure Views
6-11
Synthesizer Views Supporting Operation 6-1
Section 1 – Non-Cycle Operational Support Views
:
Topics Covered This chapter describes the Synthesizer Window views used to support instrument
operation and covers the following topics:
Topic
Manual Control View
See page
6-3
Description of View
6-3
Types of Functions
6-4
Viewing a Function Valve List/Creating a User Function
6-4
Creating a User Function
6-5
Printing Functions
6-5
Power Fail View
6-6
Purpose of View
6-6
Instrument Test View
6-7
Tests Performed
6-7
Activating Tests
6-7
Interpreting Tests
B+ Tet Calibration
6-7
6-9
Contents of View
6-9
Reagent Utilization Table
6-10
Contents of View
6-10
6-2 Synthesizer Views Supporting Operation
Manual Control View
Description of View The Manual Control view is used to exercise manual control over existing functions
and create or edit user functions. When first selected, the Manual Control view
appears as shown below:
Ready
Two kinds of functions are used in the instrument, system functions and user
functions:
♦
System functions are permanent dedicated functions provided with the
synthesizer and cannot be edited.
♦
User functions, listed in the table below, can be both viewed and edited.
Selecting a user function in the Function List (shown below) enables the
CHOOSE VALVE TO ADD scroll box enabling entry.
Table 5-4 User Functions
110
150
227
228
380-394
401-500
Synthesizer Views Supporting Operation 6-3
Types of Functions Functions are of several types:
♦
Valve functions
♦
Non-valve hardware functions
♦
Logical or cycle directive functions
Valve functions, for example, simultaneously open a series of valves to perform a
defined action during synthesis when executed from a cycle. The valves opened as
part of a function remain open for the time specified in the cycle step or otherwise as
controlled by software. See Appendix A for a complete listing and discussion of
functions.
Areas of the Manual The Manual Control view provides three scrollable lists and a fourth area which allows
Control View turning on a function:
♦
The FUNCTION LIST indicates which function is currently displayed.
♦
The VALVES OPEN FOR FUNCTION list contains a valve listing for the current
function, if that function opens valves.
♦
The CHOOSE VALVE TO ADD buttons are used to create user functions and
contains a list of valves available for creating a new function.
♦
The fourth area enables manual control and is described in the procedure
immediately below.
Exercising Manual The area on the upper left labeled Manual Control, allows you to turn on a selected
Control of a function for a specified period of time and provides the option of using the liquid
Function sensors with reagents delivered by the function. To use this portion of the table, follow
these steps:
Step
Action
1
Select the function to be turned on in the Function List.
2
Enter the time during which the function operates in the Time entry field (optional
for certain functions).
3
For functions which require a Misc entry, such as Functions 301–310 (Set Pres
Reg), enter the appropriate value in this field.
4
Click Start to turn on the selected function for the specified time, if applicable.
5
If you want to stop the function before the time specified has elapsed, click Stop.
Viewing a Function To view the valve list associated with an existing function:
Valve List ♦ Select the function to be viewed in the FUNCTION LIST, which contains a
complete list of functions contained in the instrument.
Selecting a function on this list displays the list of associated valve openings (no
valves are used in some functions) in the list entitled VALVES OPEN FOR FUNCTION
and lists the number and name of the selected function in the two fields above the list
(Function and Name).
6-4 Synthesizer Views Supporting Operation
Creating a User To create a user function, do the following:
Function
Step
1
Action
Select any of the functions in the list entitled “User Fxn” (see available numbers in
the table on page 6-3) or functions in the range 401–500 (for user functions tied to
sensors). (The function number will appear in the Function field and the Name field
will become available for entry.)
Note
Selecting an empty user function displays the function number in the
Function field and the function name in the Name field. Nothing is listed in the
VALVES OPEN list for an empty function or a non-value function.
2
Type a name for the new user function into the Name field.
3
Select the first valve to be added from the CHOOSE VALVE TO ADD list and then
click the Add button to add the valve to the VALVES OPEN FOR FUNCTION list.
The CHOOSE VALVE TO ADD list is a complete list of valves used to create user
functions.
4
Continue by selecting each valve to be added to the function in the correct order
and then clicking the Add button. If you make an error, click the Remove button.
Printing Functions The Manual Control View is one of several views from which printing can be done
(others are the Edit Cycle views, the Edit Procedure views, and the Instrument Test
view). As shown in the figure below, the print dialog box for the Manual Control view
allows two main options, printing of function text or printing of the current Manual
Control view window image.
If you choose the Text option, you can choose to print a single selected function,
system or user, or choose to print all user functions. The function number and name
are printed for all functions but valve numbers are printed only for system or user
functions with associated valves. Functions with no associated valves (system or
empty user functions) will have “None” printed for VALVES.
Note
The User functions available are listed in the table on page 6-4.
Synthesizer Views Supporting Operation 6-5
Power Fail View
Purpose of View The Power Fail view, shown below, is used to review the power failure history of the
synthesizer.
6-6 Synthesizer Views Supporting Operation
Instrument Test View
The Instrument Test view pictured below is primarily intended for Service Engineer
use. All tests in this view may be performed by the customer but interpretation of time,
current, and valve status values is “service only.” If an explanation of these values
becomes necessary, please ask your local field representative for interpretation.
Sensor status is intended for use by the customer and the service engineer. Once a
sensor calibration is performed (in the Misc Procedures view), gas and liquid values
are assigned for each of the twenty-three (there is no sensor #13) liquid sensors. The
liquid value must be at least twice the gas value for each position in order for the
calibration procedure to pass and for a Synthesis to start. Your local engineer or
technical support representative may ask you to read off the sensor values when
troubleshooting failed deliveries.
Note
The Instrument Test View is one of several views from which printing can be done
(others are the Edit Cycle views, the Edit Procedure views, and the Manual Control View.
Synthesizer Views Supporting Operation 6-7
B+ Tet Calibration
Contents of View The figure below contains the initial B+ Tet calibration values for your 3948 instrument.
If this table is deleted or otherwise damaged in your instrument, it can be restored in
one of two ways:
♦
Open the off-line copy of the database and then download the database using the
Send to Synthesizer command.
♦
Open the off-line copy of the database and then use the opened database as a
reference while you manually type in appropriate values.
Manual entry is made one field at time into the table in the ABI™ 3948 System
Control application. Select each table field, i.e. B+TET to Col A, in a particular
column and then enter the appropriate value in the time entry field at the bottom of
the table.
The values provided in this table are used by the B+Tet function in the Synthesis
module to provide a maximum time for delivering amidite from a bottle to a column
position. For example, the Tet to Col value is the time (in seconds) for the washout with
tetrazole of the synthesis valve block to ensure that there is no crossover amidite
delivery between columns.
For more information on how the B+Tet function works, see the information in
Appendix B, ABI 3948 Special Functions, under “1 B+TET to Syns” on page B-5.
Note
The CPL (Coupling) Wait Time in the cycle waits between pushes during the coupling
phase of synthesis.
6-8 Synthesizer Views Supporting Operation
Reagent Utilization Table View
Contents of View The figure below contains the initial Reagent utilization values for your ABI™ 3948
instrument. If this table is deleted or otherwise damaged in your instrument, it can be
restored in one of two ways:
♦
Open the off-line copy of the database and then download the database using the
Send to Synthesizer command.
♦
Open the off-line copy of the database and then use the opened database as a
reference while you manually type in appropriate values.
Manual entry is made one field at time into the table in the ABI™ 3948 System
Control application. Select each table field in a particular column and then enter
the appropriate value in the volume entry field at the bottom of the table.
The values provided by this table are used to calibrate the Bottle Usage View so that
the latter view can correctly indicate the amounts of reagents needed for the next run.
The volume values in the four columns are described in the following table:
Column
Description
Base
Volume consumed per addition of the specified base (from
Positions AGCT and 5–8).
Cycle
Volume consumed per synthesis cycle of specified reagents other
than amidites.
Example: for a 20 mer oligo, total TCA consumption with be
(20 x value in Cycle column) or
20 x 0.58 = 116 mL for that oligo.
Synthesizer Views Supporting Operation 6-9
Column
Description
Oligo
Volume consumed of specified reagent on a per-oligo basis.
Example: for a run of 36 purified oligos,
total consumption of TEAA will be
1.01 x 36 = 36.36 mL for the entire run (1.01 mL per oligo).
Run
6-10 Synthesizer Views Supporting Operation
Consumption of reagents used only once per run.
Section 2 – Editing Cycle and Procedure Views
Topics Covered This section contains the following topics:
Edit Cycle and Procedure Interface
6-12
Seven Editing Views
6-12
Interface Elements
6-12
Same General Layout
6-12
Cycle and Procedure Pop-Up Menus
6-13
Buttons
6-14
Entry Fields
6-16
Edit Synthesis Cycle View
Purpose and Contents of View
Edit Cleavage Cycle View
Purpose and Contents of View
Edit Purification Cycle View
Purpose and Contents of View
Edit Begin Procedure View
Purpose and Contents of View
Edit End Procedure View
Purpose and Contents of View
Edit Bottle Procedure View
6-17
6-17
6-18
6-18
6-19
6-19
6-20
6-20
6-21
6-21
6-22
Purpose and Contents of View
6-22
Misc (Miscellaneous) Procedures View
6-23
Purpose and Contents of View
6-23
Synthesizer Views Supporting Operation 6-11
Edit Cycle and Procedure Interface
Seven Editing Views Seven views are available for editing existing procedures or cycles and creating new
procedures or cycles:
♦
Edit Synthesis Cycle
♦
Edit Cleavage Cycle
♦
Edit Purification Cycle
♦
Edit Begin Procedure
♦
Edit End Procedure
♦
Edit Bottle Procedure
♦
Misc (Miscellaneous) Procedures
Interface Elements All seven editing views share the same interface elements:
♦
Pop-up menus
♦
Scroll boxes
♦
Buttons
♦
Entry fields, and
For a detailed procedure on how to use Edit views, see Section 6 in the ABI™User’s
Manual (Advanced Use of 3948 System Control).
Note
The Edit Cycle, Edit Procedure, Manual Control, and Instrument Test views are the
only views which can be printed.
Same General All Edit views have the same general layout as that shown for the Edit Cycle view
Layout example below. Refer to this figure in following the descriptions of the editor interface
which follow:
,
dy
Ready
x.xx
x.xx
6-12 Synthesizer Views Supporting Operation
Cycle and Procedure A pop-up menu displays available cycles or procedures to edit and presents the
Pop-Up Menus choices available for the particular view. For example, the Cycle menu on the Edit
Synthesis Cycle view initially presents the pop-up menu shown below:
When you select one of the names on this type of menu, the contents of the cycle or
procedure are listed in the main scroll box and the name is presented in the Name
entry field. If you select a name representing a new cycle or procedure, the main scroll
box initially has only Begin and End steps as shown in the figure below:
Synthesizer Views Supporting Operation 6-13
Buttons The following buttons are described in this subsection:
♦
Apply
♦
Copy from
♦
Execute
♦
Insert
♦
Safe/Sensor
Apply Button
Clicking the Apply Button updates the cycle with all the information entered into the
previous fields. If there is an invalid entry, the Apply button will be grayed out until all
the fields are correct.
Copy from Button
Once an empty cycle/procedure is selected, the Copy from Button becomes available:
♦
This button displays a dialog box like that shown below:
♦
Use the dialog box to copy any existing cycle from any open database as a
template for creating a new cycle or procedure.
Initially, the dialog box shows only the standard cycle provided.
Dbase 4.20, 2.20: Syn v4.20
Note
If you access a database containing several types of cycles or procedures, the dialog
box shown above will only show the cycles or procedures which are relevant for the current type
of edit view.
Execute Button
You can execute Begin, End, Bottle, and Shutdown procedures whenever the
instrument is “Ready”. The button is grayed out to disable execution when the
instrument is in any other mode.
In the Edit views for Synthesis, Cleavage/ deprotection, and Purification cycles, the
Execute button is always grayed out to indicate that you cannot manually execute
these cycles. The cycles are executed by the chemistry protocol assigned for
instrument operation.
6-14 Synthesizer Views Supporting Operation
Insert Button
This button is used to insert new blank steps into the cycle listed to the right.
Step
Action
1
Select the step above your new step.
2
Click Insert.
As soon as Insert is clicked, a new blank line is added to the cycle or procedure
listing and the cursor moves to the Function entry field
3
Select the function in one of two ways:
♦
Type in the function number in the Function field.
♦
Click the function you want to insert from the Function scroll box, shown below:
Note
Clicking a function is often easier than typing in the function number.
As soon as a function is added as a step in the main scroll box, the new
step is open for input using the remaining two entry fields, Time and Misc,
and the two radio buttons, Safe and Sensor.
Safe/Sensor Buttons
These radio buttons allow you to designate, respectively, that a new step is not a safe
step and that sensors are used for the step. (As defaults, a step is automatically
designated as a safe step which does not use sensors.) Entries made with these
buttons appear in the SAFE and SNS columns of the cycle table.
Only certain functions use liquid sensors as part of their operation.
Synthesizer Views Supporting Operation 6-15
Entry Fields The following entry fields are described in this subsection:
♦
Function Entry field (Function Scroll Box)
♦
Misc Data Entry field
♦
Name Entry field
♦
Time Entry field
Function Entry Field/Function Scroll Box
The Function Entry field is used to add a function to new blank steps placed in the
cycle or procedure in the main scroll box with the Insert Button. It requires that you
know the associated number by heart.
The Function scroll box, shown in the figure on page 6-15, is an easier way to add a
function to new blank steps since you can scroll until you identify the proper function.
Misc Data Entry Field
For some types of functions, an entry is required for this field to make an entry in the
MISC column of the cycle table. For example, a loop index or a pressure setting can
go in the MISC field. Refer to “Special Logical Functions” in Appendix A for information
on how to set parameters for special functions.
Note
If the Misc field is blank, the Apply button will remain grayed out (unavailable). All such
fields require at least a zero.
Name Entry Field
This field displays the current name of the cycle selected from the Cycle menu. It
initially has the name selected, as indicated by highlighting, allowing you to change
the name.
Time Entry Field
After you have inserted a function as a step in the cycle, you need to enter the time for
the new step in this entry field. Select the default “0.0” entry and type in the new value
to enter it into the cycle table.
6-16 Synthesizer Views Supporting Operation
Edit Synthesis Cycle View
Purpose and
Contents of View
The Edit Synthesis Cycle view allows you to produce custom cycles to perform
synthesis in the Synthesis module:
♦
The Edit Synthesis Cycle view initially appears as shown in the left side of the
figure below.
♦
The Cycle pop-up menu, shown in the right side of the figure, is used to select an
existing cycle or select a location for creating a new cycle.
,
x.xx
dy
Ready
x.xx
x.xx
The Synthesis Cycle pop-up menu can store up to 20 cycles. The first cycle is a
permanent non-programmable cycle for use in the instrument. You can copy it into one
of the other 19 locations labeled “Syn Cycle” and then edit it to create a custom cycle.
Synthesizer Views Supporting Operation 6-17
Edit Cleavage Cycle View
Purpose and The Edit Cleavage Cycle view allows you to produce custom cycles to perform
Contents of View cleavage at the Cleavage station and deprotection in the deprotection coils:
♦
The Edit Cleavage Cycle view initially appears as shown in the left side of the
figure below.
♦
The Cycle pop-up menu, shown in the right side of the figure, is used to select an
existing cycle or select a location for creating a new cycle.
,
Ready
x.xx
x.xx
x.xx
The Cleavage Cycle pop-up menu can store up to 9 cycles besides the first cycle
(which is a permanent non-programmable cycle). You can copy it into one of the other
9 locations labeled “Clv Cycle” and then edit it to create a custom cycle.
6-18 Synthesizer Views Supporting Operation
Edit Purification Cycle View
Purpose and The Edit Purification Cycle view allows you to produce custom cycles to perform
Contents of View purification at the Purification station:
♦
The Edit Purification Cycle view initially appears as shown in the left side of the
figure below.
♦
The Cycle pop-up menu, shown in the right side of the figure, is used to select an
existing cycle or select a location for creating a new cycle.
,
Ready
x.xx
x.xx
x.xx
x.xx
x.xx
The Purification Cycle pop-up menu can store up to 10 cycles. The first three cycles
are permanent non-programmable cycles for use in the instrument:
♦
The first cycle is used for purification of regular oligonucleotides.
♦
The second cycle is used for purification of dye primer oligonucleotides.
♦
The third cycle is used for purification of biotin.
You can use any of these cycles “as is” or you can copy one of them into one of the 7
locations labeled “Pur Cycle” and then edit it to create a custom cycle.
Synthesizer Views Supporting Operation 6-19
Edit Begin Procedure View
Purpose and The Begin procedure provided with the instrument is a phosphoramidite purge
Contents of View procedure that fills all phosphoramidite and tetrazole delivery lines (from the reservoir
to the reagent valve block) with fresh reagent. You should use the begin procedure
prior to beginning a run or if one of the phosphoramidite reservoirs has not been
accessed within 12 hours.
The Edit Begin Procedure View allows you to produce custom Begin procedures to
prepare for a run:
♦
The Edit Begin Procedure view initially appears as shown in the left side of the
figure below.
♦
The Cycle pop-up menu, shown in the right side of the figure below, is used to
select an existing begin procedure or select a location for creating a new
procedure.
,
Ready
x.xx
x.xx
x.xx
x.xx
The application can store up to 9 procedures besides the permanent nonprogrammable procedure provided. You can use the Begin procedure “as is” or you
can copy it into one of the other 9 locations labeled “Beg Proc” and then edit it to
create a custom procedure.
6-20 Synthesizer Views Supporting Operation
Edit End Procedure View
Purpose and An End procedure, shown below, is used to flush and clean out the system at the end
Contents of View of a run.
♦
The Edit End Procedure view initially appears as shown in the left side of the
figure below.
♦
The End Procedure popup menu, shown in the right side of the figure, is used to
select an existing cycle or select a location for creating a new cycle.
,
Ready
x.xx
x.xx
x.xx
The application can store up to 9 procedures besides the standard end procedure
provided. The standard end procedure is a permanent non-programmable cycle. You
can use this procedure “as is” or you can copy it into one of the other 9 locations
labeled “End Proc” and then edit it to create a custom procedure.
Synthesizer Views Supporting Operation 6-21
Edit Bottle Procedure View
Purpose and Bottle change procedures are used to autodilute amidites or remove empty bottles
Contents of View and replace them with bottles of fresh reagents. These procedures are used either
before beginning a synthesis or when an active synthesis has been interrupted.
Change procedures are especially important for phosphoramidite and tetrazole bottles
because they are the most sensitive to atmospheric oxygen and water.
,
Ready
)
X.XX
X.XX
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
As shown in the Procedure pop-up menu in the right side of the figure above, fifteen
non-programmable procedures are provided. The first five procedures, autodilution
procedures for amidites, include:
♦
The “AutodiluteACCT” procedure which autodilutes amidites simultaneously at all
four standard positions (positions 1 through 4)
♦
A separate autodilution procedure for each of the four standard amidite positions.
The next ten procedures, bottle change procedures, include:
♦
Procedures for use in changing the bottles at the four standard positions
(positions 1 through 4)
♦
Procedures for ammonia and tetrazole bottles
♦
Procedures for manually diluted monomers in positions 5 through 8.
The user-defined procedures, labeled “Bottle Proc.,” are for use in creating a modified
form of one of the standard procedures.
6-22 Synthesizer Views Supporting Operation
Misc (Miscellaneous) Procedures View
Purpose and This view contains various procedures needed for system maintenance or to shut
Contents of View down the instrument to prepare for long-term storage (storage for periods of one
month or longer). Procedures such as “Clean Cols” and “Clean Lines” are useful for
both maintenance or shutdown since they remove all reagents in the delivery lines and
wash and dry all chemical pathways.
One of the procedures provided with the synthesizer, “Prime Amidites”, is the initial
Misc Procedures View and is used to prime phosphoramidite delivery lines in the
instrument after using the cleaning procedures or when restoring a shut down
instrument for regular use.
,
Ready
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
x.xx
Note
Hardware Test vx.xx is a procedure used to implement hardware tests for the
Instrument Test View and can not be executed from the Misc Procedures View even though the
Execute button is active.
As you can see by examining the Cycle pop-up menu, the following types of
procedures are permanent non-programmable cycles for use in the instrument:
♦
Procedures for cleaning, priming and hardware testing
♦
Procedures for calibrating sensors, verifying sensor calibration, and setting
pressures
♦
Procedures for emptying ACN lines, and testing the sample collector and pressure
module
Note
The Janitor procedure provides more complete instrument cleanup.
You can use these procedure “as is” or you can copy one of them into an empty
procedure slot and then edit it to create a custom procedure.
Note
For information on shutting down the instrument, refer to “Shutting Down and
Restoring the Instrument” on page 5-5 of the ABI ™ 3948 System User’s Manual.
Synthesizer Views Supporting Operation 6-23
A
Valves and Functions A
In This Appendix
Topics Covered This appendix contains the basic information needed to understand ABI 3948 System
Functions, Cycles, and Procedures. For detailed information on ABI 3948 System
Special Functions, see Appendix B. For detailed information on ABI™ 3948 System
cycles and procedures, see Appendix C. For a detailed listing of ABI™3948 System
function information, see Appendix D. Appendix E provides a plumbing diagram for
use in tracing function, cycle, and procedure reagent flow paths.
This appendix covers the following topics.
Topic
See page
Information on ABI 3948 System Chemistry Terminology
A-3
Introduction
A-3
Definition of Functions, Steps, Cycles, Protocols, and Procedures
A-3
Chemistry Stages on the ABI 3948 System
A-3
Valves
A-5
List of Valves
A-5
Overview of Functions
A-7
Introduction
A-7
Function Names
A-7
Time and Misc Field Entries or Subroutines
A-7
Tracing Flow Paths for Functions
A-8
Valve Functions
A-10
Types of Valve Functions
A-10
Uses of Valve Functions
A-10
Manual Control
A-10
Creating Your Own Functions
A-10
Sensors
A-11
General Characteristics
A-11
Location of Sensors
A-11
Retries
A-12
Description of User Functions
A-12
Components of Sensor-Controlled User Functions
A-12
Names and Locations of Sensors
A-13
Valves and Functions A-1
Topic
Use of Parallel with Serial User Functions
Sensor-Controlled User Function Example
Valve Functions by Functional Category
A-13
A-14
A-15
List of Function Categories
A-15
Synthesis Valve Functions
A-15
Synthesis Column Flushing and Back Flushing Functions
A-16
Cleavage Functions
A-17
Purification and Quantitation Functions
A-18
Bottle Change Functions
A-20
User Functions
A-20
Block Functions
A-21
Non-Valve Hardware Functions
Overview
A-23
A-23
Pressure Regulating Functions
A-23
Calibration Functions
A-23
Turntable Control Functions
A-23
Miscellaneous Hardware Functions
A-24
Jaw Functions
A-24
Sample Collector Functions
A-24
Overview of Special Functions
Introduction
A-2 Valves and Functions
See page
A-25
A-25
Information on ABI 3948 System Chemistry Terminology
Introduction This section contains information essential to understanding functions, the three types
of cycles (Synthesis, Cleavage/Deprotection, and Purification) and procedures.
Automated production of oligonucleotides on the ABI™ 3948 System requires
chemical deliveries to specified destinations on the instrument. These deliveries are
controlled by electrically activating valves that open and close, creating various
pathways through the ABI™ 3948 System.
Definition of
Functions, Steps,
Cycles, Protocols,
and Procedures
Each valve is assigned a number that can be used to open or close it. Most functions
consist of a valve or set of valves that open to perform a specific delivery or task.
For example, Function 27, Cap to Syn Cols, delivers capping solution simultaneously
to all three synthesis columns. To accomplish this, Function 27 opens valves 13, 14,
15, 16, 17, 18, 22, 24, 25, and 26 simultaneously. Other functions control hardware
systems such as the turntable or control the flow of cycle execution.
A function is an individual action which may have Time and Misc (Miscellaneous)
fields. Functions can be activated manually in the Manual Control view or may be
programmed as steps in cycles (for example, Function 27 is activated for 15 seconds
in the standard synthesis cycle).
A cycle contains a series of steps that perform a chemical process. The ABI™ 3948
uses three types of cycles to produce oligonucleotides:
♦
Synthesis cycles, to add bases
♦
Cleavage/Deprotection cycles, to cleave oligonucleotides from column supports
and then deprotect them
♦
Purification cycles, to purify and quantitate or produce crude oligonucleotides for
output to the sample collector
A protocol consists of three cycles and contains all the chemical steps needed to
produce an oligonucleotide.
A Procedure is a series of steps that completes a task, such as changing a reagent
bottle. Upon command, the steps are performed once and are not repeated. There are
four types of operating procedures: begin, end, bottle and miscellaneous.
Chemistry Stages on The Synthesis, Cleavage/Deprotection, and Purification cycles are performed for each
the 3948 oligonucleotide:
Stage
Description
1
First, the Synthesis cycle synthesizes the oligonucleotides in the first row on
OneStep™ columns at the Synthesis jaw.
2
At the second stage, the turntable moves the oligonucleotide-containing columns to
the Cleavage jaw.
The Cleavage/Deprotection cycle cleaves the oligonucleotides from the OneStep
column in the jaw and transfers them to the deprotection coils to be deprotected.
3
In the third stage, the columns move to the Purification jaw where deprotection is
completed and the deprotected oligonucleotides are purified or collected as crude
product.
Valves and Functions A-3
Every time one row of columns moves to the next station, the subsequent row of
columns moves to the station just vacated. As the first row of oligonucleotides is being
cleaved at the second station, the second row of oligonucleotides is undergoing
synthesis at the first station.
When the second row in turn moves to the second station, the third row of columns
moves to the first station, and so on. This concurrent use of three stations with three
different, sequential cycles executing in parallel enables the high throughput of the ABI
3948 Synthesis/Purification system.
A-4 Valves and Functions
Valves
List of Valves During a synthesis, valves are automatically opened to create chemical pathways and
allow all necessary reagent and gas deliveries. You can also manually operate valves
in the Manual Control View before or after a synthesis or when a synthesis has been
interrupted.
Table A-1 lists all valves with brief descriptions of the associated gas or reagent flow.
Appendix D provides more detailed schematic information. Refer to “Delivery Valve
Blocks” in Chapter 2 for a physical description of the valves.
Note
To identify reagent valve blocks, see the plumbing diagram for the instrument in
Appendix D.
Table A-1 Valves with Associated Reagents and Gas Flows
Block or
Valve
Type
Valve
A
1
2
3
4
5
6
7
8
ACN to Syn
Gas to Syn
Xfr from Syn
Tetrazole
“5” amidite
“6” amidite
“7” amidite
“8” amidite
E
B
9
10
11
12
13
14
15
16
“A” amidite
“G” amidite
“C” amidite
“T” amidite
Syn Col C Lower
Syn Col B Lower
Syn Col A Lower
Xfr to Syn Low
C
17
18
19
20
21
22
23
24
25
26
27
28
D
Reagent or Gas Flow Block or
Valve
Type
Valve
Reagent or Gas Flow
Gas to Clv Lower
Xfr to Clv Lower
H2O to Clv Lower
29
30
31
32
33
34
35
36
Ammonia
Clv Col C Lower
Clv Col B Lower
Clv Col A Lower
Unused Valve 36
F
37
38
39
40
41
42
43
Gas to Dep Lower
ACN to Dep Lower
Xfr Clv to Dep
Coil A Input
Coil B Input
Coil C Input
Dep Low to Waste
NMI
Ac20
TCA
Iodine
Syn Low to Waste
G
44
45
46
47
48
49
50
Gas to Dep Upper
H2O to Dep Upper
Syn Upr to Waste
Syn Upr Hal Waste
Synthesis Col C Upper
Synthesis Col B Upper
Synthesis Col A Upper
PMS Vent
Gas to Syn Upper
H
51
52
53
54
55
Gas to Pur
ACN to Pur
TEAA
TFA
Ac Acid
Coil A Exit
Coil B Exit
Coil C Exit
Xfr from Dep
Dep Upr to Waste
Valves and Functions A-5
Table A-1 Valves with Associated Reagents and Gas Flows (continued)
Block or
Valve
Type
Valve
I
56
57
58
59
60
61
62
63
20% ACN
Xfr from Dep
Pur Col C
Pur Col B
Pur Col A
Xfr to UV
Pur Low to Waste
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
Unused Valve 64
Pur Col C Upper
Pur Col B Upper
Pur Col A Upper
Pur Upr to Waste
Gas to Pur Upper
Xfr to Hal Waste
Unused Valve 71
Waste Valve 72
Unused Valve 73
Ammonia Vent
Gas to Clv Upper
Amidite Vent
UV Lower
Gas to UV
UV to Waste
Unused Valve 80
J
A-6 Valves and Functions
Reagent or Gas Flow Block or
Valve
Type
H2O to Pur
Valve
Reagent or Gas Flow
Overview of Functions
Introduction The permanent non-programmable functions available on the instrument are considered
“System” functions to distinquish them from another function category provided on the
instrument, “User” functions. More information on User functions is provided below and
later on in this appendix.
System Functions
There are three fundamental types of functions on the ABI 3948 System:
♦
Valve functions that deliver reagents and gas throughout the system
♦
Non-valve hardware functions that control parts of the ABI 3948 System such as the
turntable, pressure regulators, or sample collector
♦
Logical or cycle directive functions such as Begin Loop, Select Pur Cols, or If Cyc
Greater.
Logical functions help to define the behavior of other functions or affect the flow of
control through the steps in a cycle
Each of the three types of functions above are covered separately as sections in this
appendix.
User Functions
Two types of User functions are provided on the instrument. One type is considered a
general purpose valve function and the other type is a special User function that can
perform sensor-controlled deliveries. Information on creating these types of User
functions is provided in Chapter 6 under “Creating a User Function” on page 6-5.
Because the sensor-controlled User functions are a special type needing separate
discussion. more information on them is provided under “Description of User Functions”
on page A-12
Function Names Functions have abbreviated names which describe the action they perform. For example,
Function 44, ACN to SynCol A, delivers acetonitrile to Column A. You can trace the
pathway created by activating Function 44 on the simplified synthesizer module
schematic shown in Figure A-1.
The third class of functions, Special Logical (Cycle Directive) functions, are functions
whose behavior cannot be anticipated by relating the function name to the instrument's
reagents or mechanical components.
Time and Misc Field In all cases, the Time and Misc fields are used as follows:
Entries or ♦ The Time field uses a time in seconds and tenths of a second.
Subroutines
♦
The Misc (Miscellaneous) field is used to declare any other type of step parameter
such as a pressure or temperature setting.
Sometimes a function's behavior is modified simply by having a non-zero instead of a zero
in the Miscellaneous field. In these cases, any non-zero value will work. ABI cycles use
999 with these functions to distinguish between “non-zero” and the specific values
required by other functions.
Valves and Functions A-7
Tracing Flow Paths To understand how the instrument performs the process of synthesis, compare the
for Functions Synthesis cycle listing provided in Appendix B and the plumbing diagram on page A-9
with the following function lists:
♦
Table A-2 on page A-15
♦
Table A-3 on page A-16
To understand the process of cleavage/deprotection, compare the
Cleavage/Deprotection cycle listing provided in Appendix B and the appropriate
portions of the synthesizer schematic in Appendix D with the following function lists:
♦
Table A-3 on page A-16
♦
Table A-4 on page A-17
♦
Table A-5 on page A-17
♦
Table A-6 on page A-18
♦
Table A-7 on page A-18
To understand the process of purification/quantitation, compare the purification cycle
listing provided in Appendix B and the appropriate portions of the synthesizer
schematic in Appendix D with the following function lists:
Table A-8 on page A-18
Table A-9 on page A-19
Table A-10 on page A-19
To achieve reagent flow, specific valves must open simultaneously so that the
following occurs:
A-8 Valves and Functions
♦
The pathway from the pressurized bottle to the valve block is opened.
♦
An exit is provided out of the valve block to waste, or to the column and then to
either the waste port or the collection vial.
Column
Valve
Block D
22
Flammable Waste
Halogenated Waste
Synthesis Col C
Synthesis Col B
23
24
25
26
Spare
(Reg 7B) Gas
27
28
Synthesis Col A
16
15
To Block C
14
13
Synthesis Col B
12
Amidite T
Amidite C
Amidite G
Amidite A
11
10
9
B
A
Synthesis Col C
C
C
8
7
6
5
4
3
2
1
Amidite 8
Amidite 7
Amidite 6
Amidite 5
Tetrazole
V 30
Gas (Reg 7C)
Reg. 4A
CH3CN
Legend
= Valve
= Bottle
CH3CN
Figure A-1
Flow path of ACN to Column A (valves 1,15,22,26)
(partial schematic of synthesis module only)
Valves and Functions A-9
Valve Functions
Types of Valve There are three basic types of valve functions:
Functions ♦ Block functions - which do not deliver to columns or coils
♦
Serial functions - which deliver to a specific column or coil and will only execute if
their designated column is active
♦
Parallel functions - which, within a given area such as cleavage, deliver to all
active columns or coils at the same time
In an instance where only a single column is active, a parallel function will behave
like the corresponding serial function dedicated to that one column.
Associated parallel and serial functions are always clustered together on the function
list in the order: Parallel Function, Serial A, Serial B, Serial C.
Uses of Valve Valve functions do the following:
Functions ♦ Direct the reagent deliveries in the Synthesis, Cleavage/Deprotection, and
Purification cycles that are necessary to produce oligonucleotides on the 3948.
♦
Constitute the following procedures: bottle change, auto-dilution, begin, end,
miscellaneous, and flow test.
These procedures as well as the three types of chemistry cycles are documented
further on in this appendix.
A valve function works by opening or closing a valve or set of valves simultaneously to
perform a specific delivery or task for a specified time, such as deliver reagents:
Reagents delivered in this way flow through the column and then to either the waste
bottle, deprotection coils, or the UV detector and the collection vial.
Manual Control Valve functions are used automatically by the system to perform chemistry or other
tasks when you run a procedure or a chemistry protocol. If you desire, you can
manually activate valves as well as other types of functions using the Manual Control
view whenever the instrument is not active.
Creating Your Own Besides activating functions which are already defined, the ABI™ 3948 System
Functions provides you with the capability of creating your own functions. As described under
“Exercising Manual Control of a Function” on page 6-4, you can create more than 100
user-defined functions in the Manual Control View and combine them with the
standard set of functions to customize procedures.
Customized procedures may also be executed from within the Edit view used to create
them (see Execute Button on page 6-14). During synthesis, the Monitor Chemistry
menu displays each function as it is activated. For information on creating your own
sensor-controlled functions, see “Description of User Functions” on page A-12.
A-10 Valves and Functions
Sensors
General Valve functions may be sensor controlled or not and work as follows:
Characteristics
Characteristic
Description
Serial and Parallel
compared to block
All serial and parallel functions work with liquid/gas sensors while
block functions do not.
Execution by time
or sensor
Sensor functions execute for the time specified in the step unless their
sensor is activated (i.e., the step field “SNS” equals YES).
In this case, execution stops either when the sensor is triggered or
when the time runs out, whichever occurs first (see the discussion
under “Retries” below).
Parallel functions
Parallel functions turn off the delivery to each column or coil
separately as each different sensor triggers.
Note
With Flush and Back Flush functions, sensors are used to detect dryness. All other
sensor functions execute until the sensor becomes wet.
Location of Sensors Sensors are located in these places:
Location
Description
Above jaw
positions
On flow paths through the jaws that clamp on the OneStep™ columns,
primarily above the columns to detect the deliveries of reagents through
the columns.
The sensors above the columns are also used to verify that columns
have been flushed dry with gas or that the cleavage or purification
vessels have been drained (back flushed) until empty.
Below synthesis
jaw positions
Between the reagent block and columns in the synthesis module to
minimize the consumption of expensive amidites.
These sensors allow the delivery of short slugs of reagent that are then
pushed into position to saturate the columns. The upper synthesis
sensors control this final push so that it is stopped at the right time to
leave these short slugs resting over the columns.
Near amidite
bottles
On flow paths out of the primary amidite bottles (A, G, C, and T —
bottles 1 through 4) to verify deliveries out of these bottles.
Near deprotection
coils
On flow paths through the three deprotection coils to detect the arrival
of samples into the coils.
Near the UV cell
On the flow path through the UV cell to detect the arrival of a sample at
the cell.
Valves and Functions A-11
Retries Retries work as listed below:
Characteristic
Description
Number of retries
Any function executing with sensor(s) active will retry a delivery or dry
up to six times if the sensor does not report success initially (except
Base+Tet).
Any function that retries more than four times will issue a message to
serve notice that trouble may be developing.
Note
Ramping functions will retry only four times instead of the
usual six and do not report retries, only failure
Execution until
time elapses or
trigger occurs
Each retry executes for as long as the time specified unless the sensor
triggers during the retry and ends the function.
Fifteen seconds
Delivery functions will wait fifteen seconds between retries to ensure
bottle repressurizing between attempts.
Back flushes and
drains
Drying functions (back flushes/drains and flushes) do not wait between
retries.
Parallel functions
Parallel functions will retry only those columns that have not already
been successful and may retry each column a different number of
times.
Description of User The last 100 functions on the ABI 3948 System function list (SynUpr Wet 401 to 500
Functions UV Dry 500) are user functions that can perform sensor-controlled deliveries. User
functions have the following characteristics:
Characteristic
Definition
Valve lists defined
by operator
Like standard user functions, the valve list for these functions can be
defined by the operator.
Sensors are
pre-assigned
These functions offer the additional advantage of having sensors
pre-assigned to them.
Engaged by YES in
SNS field
Sensors are engaged in the usual fashion by setting the SNS field in
the step to YES.
The particular sensor or set of sensors tied to a sensor user function is specified in the
default function name. As with standard user functions, the function name may be
edited as well as the valve list.
Components of There are three components to the default names provided for sensor user functions:
Sensor User
Function Names Components Description
A-12 Valves and Functions
1
The first part of the default name identifies the sensor or sensors
associated with the function.
2
The second component spells out whether the function is ‘wet’ or
‘dry,’ that is, whether the function's sensor(s) trip upon detecting
wetness (liquid delivery) or dryness (gas delivery, flush or back
flush).
3
The final component of the default function name, in keeping with
the convention for standard user functions, is the function number.
Names and Sensors have the following names and locations:
Locations of Sensors
Sensor
Name/Number
Location
SynUpr 1, 2, 3
Sensors with these designations are upper synthesis sensors.
SynMan 22, 23, 24 Sensors with these designations are synthesis manifold sensors.
SynLow 4, 5, 6
Sensors with these designations are lower synthesis sensors.
Clv 10, 11, 12
Sensors with these designations are cleavage sensors.
Coil 10, 11, 12
Sensors with these designations are coil sensors.
Pur 14, 15, 16
Sensors with these designations are purification sensors.
UV 21
This is the UV sensor.
See the Plumbing Diagram in Appendix D of this manual to verify which sensors are
associated with which columns. In some instances, the column alphabetic order and
sensor numeric order are reversed.
Use of Parallel with As with other sensor functions, parallel sensor user functions are always grouped on
Serial Sensor User the function list together with their associated serial functions. So SynUpr Wet 401 is
Functions followed by SynUprWet A 402, SynUprWet B 403, and SynUprWet C 404.
This grouping of sensor functions into sets is crucial to the operation of parallel user
functions:
Characteristic
Description
Parallel use
serial function
valve lists
Parallel sensor-controlled functions do not use their own valve lists to
execute, but use the valve lists of their associated serial functions instead.
Parallel turn on
only valves for
active columns
Parallel sensor-controlled functions turn on only the valves for active
columns by merging together the valve list for each active column's
associated serial function and excluding the valve lists of functions
associated with inactive columns.
Valves for each
column act
independently
When one column finishes its delivery before the others, its single set of
valves is deactivated. Any other active columns' valves will remain on until
their sensors trigger.
User definable functions operate according to the same scheme as follows.
♦
To program the valve list for a parallel user function, enter the appropriate valve
lists for each associated serial function.
♦
To aid in documenting the valve assignments made for serial functions associated
with a parallel user function, enter the complete three-column valve list for the
serial functions into the listing for the parallel function.
The entire valve list may then be viewed under one function, the parallel function,
in Manual Control.
Valves and Functions A-13
Sensor-Controlled As explained above in the discussion of parallel user functions, sensor-controlled
User Function parallel user functions execute the valve list settings made in the associated serial
Example functions. This means that sensor-controlled user functions are always used in groups
of four functions, the parallel function for the group and the three serial functions
associated with it.
For example, a user might want to deliver Tetrazole (TET) until it tripped the upper
sensors (the sensors above the synthesis columns) instead of the manifold sensors as
the standard TET to SynCols (Fxn 48) parallel function would do. This can be done
using these four functions: SynUpr Wet 401, SynUpr Wet A 402, SynUpr Wet B 403,
and SynUpr Wet 404.
Using these functions requires the following four steps:
Step
Action
1
Put the desired valve lists into the associated serial functions.
2
Open the cycle intended to execute the parallel function and turn the sensors on for
the parallel function (“Yes” in the SNS field).
3
Set a trip time (value in seconds in the TIME field) greater than required for tripping
sensors (perhaps 26 seconds).
4
Run the cycle in which you called the parallel function to execute it.
The following valve list entries are required for the functions in this example:
Function
Valves
SynUpr Wet A 402
Valve 3 (Tetrazole)
Valve 15 (Column A)
SynUpr Wet B 403
Valve 3 (Tetrazole)
Valve 14 (Column B)
SynUpr Wet C 404
Valve 3 (Tetrazole)
Valve 13 (Column C)
Note
The value set for trip time can be any value greater than actually needed for the
sensor(s) to trip. The actual trip values will be reported to the Microphone file.
A-14 Valves and Functions
Valve Functions by Functional Category
List of Function Valve functions are listed by functional category within this section. The third column
Categories of the function table for each category is used to indicate the type of Valve function (a
blank, no entry, indicates a Serial function, Parallel indicates a Parallel Function,
Ramping indicates a Ramping Function, and Block indicates a Block Function.
Note
The functions in this category are used together with Non-valve Hardware functions
and Logical or Cycle Directive functions to perform instrument chemistry.
To understand how valve functions work in a particular chemistry cycle, refer to the
chemistry cycle listing in Appendix B and the material entitled “Special Functions” in
this appendix to understand how the instrument performs chemistry. The Plumbing
Diagram is provided in Appendix D to aid you in tracing out chemistry flow paths.
This section categorizes functions in the following areas:
♦
Synthesis Valve Functions
♦
Synthesis Column Flushing and Back Flushing Functions
♦
Cleavage Functions
♦
Purification Functions
♦
Bottle Change Functions
♦
User Functions
♦
Purification and Quantitation Functions
♦
Block Functions
Synthesis Valve Table A-2 shows the Synthesis functions used for reagent deliveries to columns in
Functions synthesis jaws.
Table A-2 Synthesis Functions
Func.
Number
Function Name
Parallel/Block/ Func.
Special Func. Number
1
B+ TET to Syns
2
A+ TET to Syn A
3
G+ TET to Syn A
19
G+ TET to Syn C
4
C+ TET to Syn A
20
C+ TET to Syn C
5
T+ TET to Syn A
21
T+ TET to Syn C
6
5+ TET to Syn A
22
5+ TET to Syn C
7
6+ TET to Syn A
23
6+ TET to Syn C
8
7+ TET to Syn A
24
7+ TET to Syn C
9
8+ TET to Syn A
25
8+ TET to Syn C
10
A+ TET to Syn B
26
Coupling Wait
11
G+ TET to Syn B
27
CAP to Syn Cols
12
C+ TET to Syn B
28
CAP to SynCol A
13
T+ TET to Syn B
29
CAP to SynCol B
14
5+ TET to Syn B
30
CAP to SynCol C
See “Special”
functions.
Function Name
17
8+ TET to Syn B
18
A+ TET to SynC
Parallel/Block/
Special Func.
See “Special”
functions
Parallel
Valves and Functions A-15
Table A-2 Synthesis Functions (continued)
Func.
Number
Function Name
Parallel/Block/ Func.
Special Func. Number
Function Name
Parallel/Block/
Special Func.
Parallel
15
6+ TET to Syn B
33
IODINE to Syns
16
7+ TET to Syn B
34
IODINE to Syn A
37
IODINE to Waste
Block
45
ACN to SynCol B
38
TCA to Syn Cols
Parallel
46
ACN to SynCol C
39
TCA to SynCol A
47
ACN to SynWaste
40
TCA to SynCol B
48
TET to Syn Cols
41
TCA to SynCol C
49
TET to SynCol A
43
ACN to Syn Cols
50
TET to SynCol B
44
ACN to SynCol A
51
TET to SynCol C
Parallel
Block
Parallel
Synthesis valve functions provide for flow of reagents (driven by argon pressure) from
the reservoirs, through valve blocks A, B, or C, through the OneStep columns, where
all chemical reactions necessary for synthesis occur.
Two other types of functions, Flushing and Back Flushing, although they do not deliver
reagents, are essential to Synthesis. These functions perform tasks such as flushing
and back flushing columns (see Table A-3 below).
Synthesis Column The functions in Table A-3 flush and back flush synthesis columns:
Flushing and Back
Table A-3 Synth-Gas Flushes and Back Flushes
Flushing Functions
Function
Number
A-16 Valves and Functions
Function Name
53
Flush Syn Cols
54
Flush Syn Col A
55
Flush Syn Col B
56
Flush Syn Col C
57
Back Flush Syns
58
Back Flush Syn A
59
Back Flush Syn B
60
Back Flush Syn C
61
Trityl Flush Syn
62
TritylFlsh SynA
63
TritylFlsh SynB
64
TritylFlsh SynC
65
SynLowBlk Flush
Parallel/Block/
Special Function
Parallel
Parallel
Block
Block
Cleavage Functions The following tasks are performed by functions listed in this section:
♦
Delivery to Clv Cols (Table A-4)
♦
Gas Flushes and Pressurization (Table A-5)
♦
Oligonucleotide Transfer (Table A-6)
♦
Water Deliver to Deprotection Coil (Table A-7)
Delivery to Cleavage Columns
The functions listed in Table A-4 prime delivery lines and deliver reagents to cleavage
columns.
Table A-4 Functions for Delivery of Reagents to Clv Cols
Func.
Number
Function Name
111
Ammonia to Clvs
112
Parallel/Block/ Func.
Special Func. Number
Function Name
Parallel/Block/
Special Function
126
H2O to Clv Cols
Parallel
Ammonia to ClvA
127
H2O to ClvCol A
113
Ammonia to ClvB
128
H2O to ClvCol B
114
Ammonia to ClvC
129
H2O to ClvCol C
115
Ammonia toWaste
130
H2O to ClvWaste
116
ACN to Clv Cols
117
ACN to Clv A
118
ACN to Clv B
119
ACN to Clv C
120
ACN to ClvWaste
Parallel
Block
Block
Parallel
Block
Gas Flushes and Pressurization
The functions listed in Table A-5 flush and back flush cleavage columns and
deprotection coils:
Table A-5 Functions for Gas Flushes and Pressurization
Func.
Number
Function Name
Parallel/Block/ Func.
Special Func. Number
121
Gas to Clv Cols
134
Back Flsh Clv C
122
Gas to ClvCol A
138
Press Line
Block
123
Gas to ClvCol B
144
Clv Blk Flush
Block
124
Gas to ClvCol C
153
DepLowBlk Flush
Block
125
Gas to Clv II
Block
155
Gas toDep Coils
Parallel
131
Back Flush Clvs
Parallel
156
Gas to Coil A
132
Back Flsh Clv A
157
Gas to Coil B
133
Back Flsh Clv B
158
Gas to Coil C
Parallel
Function Name
Parallel/Block/
Special Function
Valves and Functions A-17
Oligonucleotide Transfer
The functions listed in Table A-6 transfer oligonucleotides from the cleavage columns
into the deprotection coils.
Table A-6 Functions for Oligonucleotide Transfer
Func.
Number Function Name
Parallel/Block/ Func.
Parallel/Block/
Special Func. Number Function Name Special Function
141
Xfer Clv Col A
153
DepLowBlk Flush
Block
142
Xfer Clv Col B
154
Dep Upr Blk
Rinse
Block
143
Xfer Clv Col C
166
Xfr Coil A
Ramping
151
Dep Xfer Rinse
Block
167
Xfr Coil B
Ramping
152
Dep Xfer Flush
Block
168
Xfr Coil C
Ramping
Water Delivery to Deprotection Coil
The functions listed in Table A-7 deliver water to the deprotection coils:
Table A-7 Functions to Deliver Water to Deprotection Coils
Function
Number
Function Name
163
H2O to Coil A
164
H2O to Coil B
165
H2O to Coil C
Purification and The following tasks are performed by functions listed in this section:
Quantitation ♦ Delivering Reagents to Purification Columns/UV Detector (Table A-8)
Functions
♦
Rinsing and Flushing Deprotection Columns (Table A-9)
♦
Collecting and Quantitating Samples (Table A-10)
Delivery of Reagents to Purification Columns/UV Detector
The functions listed in Table A-8 deliver reagents to the purification columns and the
UV detector.
Table A-8 Functions Delivering Reagents to Pur. Columns/UV Detector
Func.
Number Function Name
A-18 Valves and Functions
173
ACN to Pur Cols
174
Parallel/Block/ Func.
Special Func. Number Function Name
Parallel
190
TFA to PurCol B
ACN to Pur ColA
191
TFA to PurCol C
175
ACN to Pur ColB
193
20%ACN to Purs
176
ACN to Pur ColC
194
20%ACN to Pur A
177
ACN to PurWaste
Block
195
20%ACN to Pur B
178
TEAA to PurCols
Parallel
196
20%ACN to Pur C
179
TEAA to Pur A
197
20%ACN to Waste
180
TEAA to Pur B
198
AcAcid to Purs
181
TEAA to Pur C
199
AcAcid to Pur A
182
TEAA to Waste
200
AcAcid to Pur B
Block
Parallel/Block/
Special Func.
Parallel
Parallel
Table A-8 Functions Delivering Reagents to Pur. Columns/UV Detector (continued)
Func.
Number Function Name
183
H2O to Pur Cols
184
Parallel/Block/ Func.
Special Func. Number Function Name
Parallel
201
AcAcid to Pur C
H2O to PurCol A
202
AcAcid to Waste
185
H2O to PurCol B
211
Pur A to UV
186
H2O to PurCol C
212
Pur B to UV
188
TFA to Pur Cols
213
Pur C to UV
189
TFA to PurCol A
214
H2O to UV
Parallel
Parallel/Block/
Special Func.
Block
Block
Rinsing and Flushing Deprotection Columns
The functions in Table A-9 rinse and flush the deprotection columns:
Table A-9 Functions for Rinsing and Flushing Deprotection Columns
Func.
Number Function Name
171
Pur Xfer Rinse
172
Pur Xfer Flush
203
Gas to Pur Cols
204
Parallel/Block/ Func.
Special Func. Number Function Name
Parallel/Block/
Special Func.
Block
208
Back Flsh Pur A
Block
209
Back Flsh Pur B
Parallel
210
Back Flsh Pur C
Gas to PurCol A
218
PurLowBlk Flush
Block
205
Gas to PurCol B
219
PurUprBlk Flush
Block
206
Gas to PurCol C
220
Pur Vent
Block
207
Back Flush Purs
222
SC Block Flush
Block
Parallel
Collecting and Quantitating Samples
The functions listed in Table A-10 collect and quantitate samples:
Table A-10 Functions for Collecting and Quantitating Samples
Function
Number
Function Name
Parallel/Block/
Special Function
135
Crude A to UV
Ramping
136
Crude B to UV
Ramping
137
Crude C to UV
Ramping
211
Pur A to UV
Ramping
212
Pur B to UV
Ramping
213
Pur C to UV
Ramping
215
Xfer UV to SC
Block
217
Flsh UV toWaste
Block
Valves and Functions A-19
Bottle Change The functions listed in Table A-11 are used during bottle change procedures:
Functions
Table A-11 Bottle Change Functions
Func.
Number
Function Name
Func.
Number
Function Name
82
ACN AMIDITE A
95
Mix AMIDITE 5
83
ACN AMIDITE G
96
Mix AMIDITE 6
84
ACN AMIDITE C
97
Mix AMIDITE 7
85
ACN AMIDITE T
98
Mix AMIDITE 8
86
ACN AMIDITE 5
87
Mix AG
87
ACN AMIDITE 6
98
Mix AGCT
88
ACN AMIDITE 7
99
Mix AGCT
89
ACN AMIDITE 8
100
Mix AG
90
Mix AMIDITE A
101
Mix CT
91
Mix AMIDITE G
102
AMIDITE Vent
92
Mix AMIDITE C
103
Ammonia Vent
93
Mix AMIDITE T
104
Mix Ammonia
User Functions User Functions are a reserved class of valve function which enables the user to define
functions using any valve listed in the Manual Control View. The empty functions listed
in Table A-12 are available for user definition:
Note
The functions in the range 401–500 are discussed under “Description of User
Functions” on page A-12.
Table A-12 Functions for User Definition (User Functions)
A-20 Valves and Functions
150
383
389
380
386
392
227
384
390
381
387
393
228
385
391
382
388
401-500
Block Functions Block functions are functions which do not deliver to column or deprotection coils and
include functions which perform the following tasks:
♦
Priming Delivery Lines - below
♦
Priming Functions for Cleavage Columns - below
♦
Priming Functions for Purification Columns - on page A-22
♦
Gas Flush, Pressurization, and Rinse - on page A-22
♦
Venting Functions for Block and Reagent Bottles - on page A-22
Priming Delivery Lines
The functions in Table A-13 are those used to prime delivery lines.
Table A-13 Functions for Priming Delivery Lines
Function
Number
Function Name
Function
Number
Function Name
31
AC2O to Waste
115
Ammonia toWaste
32
NMI to Waste
120
ACN to ClvWaste
37
IODINE to Waste
42
TCA to Trityl
47
ACN to SynWaste
52
TET to Waste
74
A AMIDITE Waste
75
G AMIDITE Waste
76
C AMIDITE Waste
77
T AMIDITE Waste
78
5 AMIDITE Waste
79
6 AMIDITE Waste
80
7 AMIDITE Waste
81
8 AMIDITE Waste
Priming Functions for Cleavage Columns
The functions in Table A-14 prime cleavage columns:
Table A-14 Functions for Priming Clv Cols
Function
Number
Function Name
115
Ammonia to Waste
120
ACN to ClvWaste
130
H2O to ClvWaste
Valves and Functions A-21
Priming Functions for Purification Columns
The functions in Table A-15 prime purification columns:
Table A-15 Functions for Priming Purification Columns
177
ACN to PurWaste
182
TEAA to Waste
187
H2O to PurWaste
192
TFA to Waste
197
20%ACN to Waste
202
ACAcid to Waste
Gas Flush, Pressurization, and Rinse Functions
The functions in Table A-16 are used to flush, back flush, pressurize valve blocks and
various system components, and perform block rinses:
Table A-16 Functions for Gas Flushes, Pressurization, and Rinses
Function
Number
Function Name
Function
Number
Function Name
65
SynLowBlk Flush
132
Back Flush Clv A
105
SynUprBlk Flush
133
Back Flush Clv B
106
LowTrityl Flush
134
Back Flush Clv C
107
UprTrityl Flush
138
Press Line
122
Gas to ClvColA
139
Compress
123
Gas to ClvColB
140
Clv Vent
124
Gas to ClvColC
151
Dep Xfer Rinse
125
Gas to Clv II
144
Clv Blk Flush
132
Back Flush Clv A
152
Dep Xfer Flush
133
Back Flush Clv B
153
DepLowBlk Flush
134
Back Flush Clv C
154
DepUprBlk Rinse
138
Press Line
171
Pur Xfer Rinse
139
Compress
172
Pur Xfer Flush
140
Clv Vent
218
PurLowBlk Flush
151
Dep Xfer Rinse
219
PurUprBlk Flush
144
Clv Blk Flush
221
DepUprBlk Flush
152
Dep Xfer Flush
222
SC Block Flush
Venting Functions for Block and Reagent Bottles
The functions in Table A-17 are used to vent various blocks and reagent bottles:
Table A-17 Functions for Block Venting
A-22 Valves and Functions
Function
Number
Function Name
Function
Number
66
Syn Upper Vent
101
AMIDITE Vent
67
Syn Lower Vent
102
Ammonia Vent
Function Name
Non-Valve Hardware Functions
Overview Non-valve hardware functions encompass all hardware related functions except for
valves and include the following:
♦
Pressure Regulating Functions
♦
Calibration Functions
♦
Turntable Control Functions
♦
Miscellaneous Functions (UV, Relay, Heater/Fan, Toggle)
♦
Jaw Functions
♦
Sample Collector Functions
Pressure Regulating The functions in Table A-18 regulate system pressures:
Functions
Table A-18 Functions to Regulate Pressure
Function
Number
Function Name
Function
Number
Function Name
301
Set Pres Reg 1
309
Set Pres Reg 9
302
Set Pres Reg 2
310
Set Pres Reg 10
303
Set Pres Reg 3
311
Set Press RegAll
304
Set Pres Reg 4
312
Press Reg On
305
Set Pres Reg 5
313
Press Reg Off
306
Set Pres Reg 6
314
Pres Reg AllOn
307
Set Pres Reg 7
315
Pres Reg All Off
Calibration The functions in Table A-19 calibrate system pressure as well as gas and liquid
Functions sensors:
Table A-19 Functions to Calibrate Pressure and Sensors
Function
Number
Function Name
Function
Number
Function Name
320
Check Snsr Cal
323
Calib AllGasSns
321
CalibrateGasSns
324
Calib AllLiqSns
322
CalibrateLiqSns
Turntable Control The functions in Table A-20 control the turntable:
Functions
Table A-20 Turntable Control Functions
Function
Number
Function Name
239
TurnTable Home
240
TurnTable Next
241
TurnTable Prev
Valves and Functions A-23
Miscellaneous The functions in Table A-21 control relays, fan, heater, Set Temperature, and Toggle
Hardware Functions Valve functions:
Table A-21 Hardware Control Functions
Function
Number
Function Name
Function
Number
Function Name
216
UV Reading
170
Start Depro Htr
223
Set UV Scale
255
Fan On
249
Relay 1 On
256
Fan Off
250
Relay 1 Off
257
Heater On
251
Relay 1 Pulse
258
Heater Off
252
Relay 2 On
260
Set Coil Temp
253
Relay 2 Off
261
Wait Coil Temp
254
Relay 2 Pulse
262
Tggle Vlves On
169
Depro Htr Wait
263
Tggle Vlves Off
Jaw Functions The functions in Table A-22 control the jaws in the three chemistry modules:
Table A-22 Jaw Functions
Function
Number
Function Name
231
Syn Jaw Open
232
Syn Jaw Close
233
Clv Jaw Open
234
Clv Jaw Close
235
Pur Jaw Open
236
Pur Jaw Close
237
Open All Jaws
238
Close All Jaws
Sample Collector The functions in Table A-23 control the sample collector:
Functions
Table A-23 Sample Collector Functions
Function
Number
A-24 Valves and Functions
Function Name
242
SC Waste
243
SC Home
243
SC Next
244
SC Open
245
SC Close
246
SC Needle Up
247
SC Needle Down
264
SC Prev
ABI 3948 System Special
Functions
B
B
In This Appendix
Topics Covered This appendix contains a detailed listing of ABI™ 3948 System Special Functions.
The appendix contains the following topics:
Topic
Overview of ABI 3948 System Special Functions
Introduction
Listing of Special Functions
See page
B-2
B-2
B-5
ABI 3948 System Special Functions B-1
Overview of Special Functions
Introduction A number of “special” functions have been developed for the ABI 3948 System. These
are functions whose behavior cannot be anticipated by relating the function name to
the instrument's reagents or mechanical components.
Often, these functions are normal sensor functions that have certain extra capabilities
built into them. Typically, these extra features are engaged by setting values in the
“MISC” parameter field of a step. All of the logical or cycle directive functions fall into
this class of special functions and usually invoke the Miscellaneous field as well.
The special functions listed in Table B-1 are discussed in order of function number in
the next subsection. Where a group of functions are all of the same type or work in
conjunction with each other, they are discussed together at the point where the first
function of the group appears on the list.
For ready reference, functions are always listed once according to their position on the
function list. This ordered listing will contain a reference to an earlier listing if the
function was discussed previously as part of a function group.
Note
Function 110 will report the database version number found in your instrument.
Table B-1 Table of Special Functions
Func.
Number
Func.
Number
Function Name
Func.
Number
B+ TET to Syns
176
ACN to PurCol C
230
Go Sub On Fail
26
Coupling Wait
179
TEAA to PurCol A
232
Syn Jaw Close
39
TCA to Syn Col A
180
TEAA to PurCol B
234
Clv Jaw Close
40
TCA to Syn Col B
181
TEAA to PurCol C
236
Pur Jaw Close
41
TCA to Syn Col C
184
H2O to PurCol A
251
Relay 1 Pulse
44
ACN to SynCol A
185
H2O to PurCol B
254
Relay 2 Pulse
45
ACN to SynCol B
186
H2O to PurCol C
259
Wait
46
ACN to SynCol C
189
TFA to PurCol A
260
Set Coil Temp
109
Waste to Trityl
190
TFA to PurCol B
261
Wait Coil Temp
110
Database v4.20
191
TFA to PurCol C
262
Tggle Vlves On
135
Crude A to UV
194
20%ACN to Pur A
263
Tggle Vlves Off
136
Crude B to UV
195
20%ACN to Pur B
265
Set Rmp Fxn Sns
137
Crude C to UV
196
20%ACN to Pur C
266
Set Rmp Fxn Trp
141
Xfer Clv Col A
199
AcAcid to Pur A
267
Set Rmp Fxn Chk
142
Xfer Clv Col B
200
AcAcid to Pur B
268
Set Rmp Fxn Dly
143
Xfer Clv Col C
201
AcAcid to Pur C
269
Send Message
166
Xfer Coil A
207
Back Flush Purs
270
Comment
167
Xfer Coil B
211
Pur A to UV
271
Begin of Cycle
168
Xfer Coil C
212
Pur B to UV
272
End of Cycle
169
Depro Htr Wait
213
Pur C to UV
273
Begin Loop
170
Start Depro Htr
216
UV Reading
274
End Loop
174
ACN to PurCol A
223
Set UV Scale
275
Start Here
175
ACN to PurCol B
229
End Row SCP/123
276
Exit DMT On
1
B-2 ABI 3948 System Special Functions
Function Name
Function Name
Table B-1 Table of Special Functions (continued)
Func.
Number
Function Name
Func.
Number
Function Name
Func.
Number
Function Name
277
If Pur Col A
407
SynUprWet B 407
447
Clv Dry B 447
278
If Pur Col B
408
SynUprWet C 408
448
Clv Dry C 448
279
If Pur Col C
409
SynUpr Wet 409
449
Clv Dry449
280
Else
410
SynUprWet A 410
450
Clv Dry A 450
281
Endif
411
SynUprWet B 411
451
Clv Dry B 451
282
Select Pur Cols
412
SynUprWet C 412
452
Clv Dry C 452
283
Select Act Cols
413
SynUpr Dry 413
453
Coil Wet 453
284
Clv Wants Depro
414
SynUprDry A 414
454
Coil Wet A 454
285
Pur Owns Depro
415
SynUprDry B 415
455
Coil Wet B 455
286
Clv Owns Depro
416
SynUprDry C 416
456
Coil Wet C 456
287
Go Sub
417
SynMan Wet 417
457
Coil Dry 457
288
Return Sub
418
SynManWet A 418
458
Coil Dry A 458
289
Sub Label
419
SynManWet B 419
459
Coil Dry B 459
290
Goto End
420
SynManWet C 420
460
Coil Dry C 460
291
If Any Act Cols
421
SynMan Wet 421
461
Pur Wet 461
292
Go Sub A
422
SynManWet A 422
462
Pur Wet 462
293
Go Sub B
423
SynManWet B 423
463
Pur Wet 463
294
Go Sub C
424
SynManWet C 424
464
Pur Wet 464
295
If First Cycle
425
SynMan Wet 425
465
Pur Wet 465
296
If Last Cycle
426
SynManWet A 426
466
Pur Wet 466
297
If Crude Col A
427
SynManWet B 427
467
Pur Wet 467
298
If Crude Col B
428
SynManWet C 428
468
Pur Wet 468
299
If Crude Col C
429
SynLow Wet 429
469
Pur Wet 469
300
Engage All Cols
430
SynLowWet A 430
470
Pur Wet A 470
316
If Cyc Greater
431
SynLowWet B 431
471
Pur Wet B 471
317
Save Reg Pres
432
SynLowWet C 432
472
Pur Wet C 472
318
Restore RegPres
433
SynLow Dry 433
473
Pur Wet 473
319
Revive Act Cols
434
SynLowDry A 434
474
Pur Wet A 474
320
Check Snsr Cal
435
SynLowDry B 435
475
Pur Wet B 475
325
Jaw Test Times
436
SynLowDry C 436
476
Pur Wet C 476
329
Begin Leak Test
437
Clv Wet 437
476
Pur Wet 477
330
End Leak Test
438
Clv Wet A 438
478
Pur Wet 478
396
Time Stamp
439
Clv Wet B 439
479
Pur Wet 479
400
* Reserved *
440
Clv Wet C 440
480
Pur Wet 480
401
SynUpr Wet 401
441
Clv Wet 441
481
Pur Dry 481
402
SynUprWet A 402
442
Clv Wet A 442
482
Pur Dry A 482
403
SynUprWet B 403
443
Clv Wet B 443
483
Pur Dry B y 483
404
SynUprWet C 404
444
Clv Wet C 444
484
Pur Dry C 484
405
SynUpr Wet 405
445
Clv Dry 445
485
Pur Dry 485
406
SynUprWet A 4 06
446
Clv Dry A 446
486
Pur Dry A 486
ABI 3948 System Special Functions B-3
Table B-1 Table of Special Functions (continued)
Func.
Number
Function Name
Func.
Number
Function Name
Func.
Number
Function Name
487
Pur Dry B 487
492
UV Wet 492
497
UV Wet 497
488
Pur Dry C 488
493
UV Wet 493
498
UV Dry 498
489
UV Wet 489
494
UV Wet 494
499
UV Dry 499
490
UV Wet 490
495
UV Wet 495
500
UV Dry 500
491
UV Wet 491
496
UV Wet 496
B-4 ABI 3948 System Special Functions
Listing of Special Functions
This subsection provides a discussion for each of the ABI™ special functions listed in
Table B-1 in the previous subsection.
Functions are listed in two ways:
♦
Individual functions are listed by number and name to the left, as is done for “1
B+Tet to Syns” below.
In some cases, the only material provided for an individual function will be a
reference to where the function is discussed as part of a group of functions.
♦
1
B+TET to
Syns
Functions discussed as a group will be listed at the point in the list where the first
function of the group appears on the list. In these cases, all the functions in the
group are listed in a table before the discussion.
This function appears to be a parallel function but in actuality it is three serial functions
combined as each base must be added to each column individually. When viewed in
Manual Control, the valve list shown is very short because the complete valve list for
this function is largely determined by two things: 1) the base being added to a
particular column and 2) which columns are currently active.
This is the only sensor function that does not have the capacity to auto-resume (see
function 232, Syn Jaw Close, et al.). This is also the only function that may check two
sensors to verify delivery to a single column: a given column's synthesis manifold
sensor is used to terminate delivery while base delivery is verified by sensors on some
amidite bottles (bottles 1 to 4 have sensors and bottles 5 to 8 do not).
For each column that is active, B+ TET to Syns execution goes through two stages:
delivering the amidite combined with tetrazole and then rinsing out the block with
tetrazole alone. With all columns active, this function must execute both stages three
times over. The B+TET Calibration Table specifies the delivery times for each stage
given any combination of bottle being delivered from and column being delivered to.
The B+TET times in this table are the maximum times allowed for the delivery sensors
to trip. The TET to Col times are fixed delivery times and vary with the number of valve
ports between the bottle being accessed and the column being delivered to.
26
Coupling Wait
This function acts as a wait step. The time for the wait does not come from the time
field, however, and any time listed in the step is ignored. The wait time is determined
by the Base + TET Calibration Table. If the various bases in the current cycle have
different coupling wait times, the longest time in the table for this set of amidites is the
wait time used by this function.
39
TCA to
SynCol A
The discussion below the table applies to all the functions listed in the table.
39
TCA to SynCol A
to
41
TCA to SynCol C
44
ACN to SynCol A
to
46
ACN to SynCol C
174
ACN to PurCol A
to
176
ACN to PurCol C
179
TEAA to PurCol A
to
181
TEAA to PurCol C
184
H2O to PurCols A
to
186
H2O to PurCols C
189
TFA to PurCols A
to
191
TFA to PurCols C
ABI 3948 System Special Functions B-5
194
20%ACN to Pur A
to
196
20%ACN to Pur C
199
AcAcid to Pur A
to
201
AcAcid to Pur C
Whenever their associated column is inactive and the Miscellaneous (MISC) field of
the step is non-zero, the functions listed above will wait instead of executing for the
allotted step time (TIME). This is useful when performing push/wait loops where
pushes are done serially and the wait time for one column is defined in whole or in
part by the time taken to push reagents to the other columns.
109
Waste to Trityl This function is used to temporarily direct waste leaving the upper synthesis block to
the trityl (halogenated) waste bottle when executing functions that would normally
deliver into the flammable waste bottle. This allows certain housekeeping steps in
synthesis to be used with both halogenated and non-halogenated reagents.
When Waste to Trityl is executed with a non-zero value in the Miscellaneous (MISC)
field, it sets up the valve controller to substitute valve 23 for valve 22 whenever valve
22 is called for by a function. Execution will not revert to normal until this function is
called with a zero in the Miscellaneous field.
110
Database
Version
135
Crude A to UV The discussion below the table applies to all the functions listed in the table.
This is a function reserved to have its name show the version number of the database
being used by the ABI 3948 System.
135
Crude A to UV
to
137
Crude C to UV
141
Xfer Clv Col A
to
143
Xfer Clv Col C
166
Xfer Coil A
to
168
Xfer Coil C
211
Pur A to UV
to
213
Pur C to UV
Collectively, the functions listed above are known as ramping functions. When
executing with sensors active and a regulator number listed in the Miscellaneous
(MISC) field, these functions will increase (ramp) the pressure on the specified
regulator by one psi on each retry. Ramping functions will retry only four times instead
of the usual six and do not report retries, only failures. When a ramping function
sensor triggers, the function waits momentarily and then rechecks the sensor to verify
the continued presence of liquid. The behavior of these functions is defined in part by
the Set Rmp Fxn functions described later (functions 265–268), which functions must
be called before these ramping functions can be used.
The Xfer Clv Col functions are a special case of ramping function. If these functions
have not been successful by the conclusion of the final ramp, a recovery will be
attempted. When a recovery is required, it is usually the result of a leaking jaw. Such a
leak can prevent the ammonia slug containing the cleaved oligo from being effectively
pushed toward the coils after it has passed through the jaws. (While the liquid slug is
moving through the jaws, it helps to hold a seal. Once it passes, gas can leak rapidly
and bleed off the pressure needed to keep the slug moving.)
The recovery deactivates the valve that delivers from the relevant cleavage vessel into
the lower cleavage block (valve 33, 34, or 35) and attempts a final retry with the lower
cleavage block pressurization valve activated instead (valve 29). The effect of this
recovery is to flush into the coils any cleaved oligo that might be caught in the transfer
B-6 ABI 3948 System Special Functions
path from the lower cleavage block to the deprotection system. Before this recovery
flush begins, the pressure is first reduced by two psi.
169
Depro Htr
Wait
For this function, the Miscellaneous (MISC) field is used to declare the minimum
number of minutes the deprotection heater must be on in order for deprotection to be
completed. The Miscellaneous field is used to declare time in whole minutes since the
maximum allowable entry in seconds (480) is not long enough to assure complete
deprotection. If the heater has not been on long enough, the function calculates the
remaining time it must wait and then waits for that period. If the cycle step calls for a
zero wait time, this function will substitute the default wait time specified by the
“Deprotect Mins” setup variable in the Instrument Preferences Window. This function
is used to guarantee complete deprotection despite any variability in cycles or
instrument performance.
170
Start Depro
Htr
This function is essentially identical to function 260, Set Coil Temp, except that it
informs the 3948 that the heater has been turned on specifically to begin deprotection.
This allows the clock for Depro Htr Wait (above) to begin counting time once the
heater has become hot. The temperature setpoint is entered in degrees C in the
Miscellaneous (MISC) field. If the cycle step calls for a zero deprotection temperature,
this function will substitute the default temperature specified by the “Deprotect Temp”
setup variable in the Instrument Preferences Window.
174
ACN to
PurCol A
The discussion below the table applies to all the functions listed in the table.
174
ACN to PurCol A
to
176
ACN to PurCol C
179
TEAA to PurCol A
to
181
TEAA to PurCol C
184
H2O to PurCol A
to
186
H2O to PurCol C
189
TFA to PurCol A
to
191
TFA to PurCol C
194
20%ACN to Pur A
to
196
20%ACN to Pur C
199
AcAcid to Pur A
to
201
AcAcid to Pur C
See preceding discussion of function 39,TCA to Syn Col A, et al.
ABI 3948 System Special Functions B-7
207
Back Flush
Purs
This function is a normal sensor function that is able to ramp regulator pressures on
retries. As usual for a sensor function, it will retry up to six times. In addition, if a
regulator number is specified in the Miscellaneous (MISC) field, this function will ramp
that regulator one psi on each retry. Unlike a true ramping function, it does not verify a
sensor trigger and the function behavior is not affected by the ramping functions (see
function 135, Crude to UV, et al.).
All Flush or Back Flush functions have the ramping capability. Since they are executed
with the initial pressure always set to the maximum, there is no point in specifying a
pressure ramp. Back Flush Purs, however, is used at a low initial pressure—but only in
one instance. This occurs during purification when a slow drain of the vessel is used in
order to best bind the deprotected oligo to the column. Ramping is specified in this
instance to ensure a complete emptying of the vessel(s) when binding.
211
See preceding discussion of function 135, Crude A to UV, et al.
213
Pur A to UV
to
Pur C to UV
216
UV Reading
A value of zero in the Miscellaneous (MISC) field instructs UV Reading to take a
baseline reading. This is generally done when the UV cell is filled with water. A value
of 1, 2, or 3 calls for the taking of an oligo quantitation reading for the A, B, or C
column, respectively. The most recent baseline reading is subtracted from the
quantitation reading and the resulting o.d.u. and pmol/mL values are logged for
reporting to the Run File at the end of the run.
223
Set UV Scale This function can be used to scale the translation of UV voltage readings into ODU.
The Miscellaneous (MISC) field is used to define the scaling factor in percent. The
scaling factor defaults to 100 without the intervention of this function. A scaling factor
less than 100 will produce lower ODU readings and a factor greater than 100 will
amplify the ODU values reported. Any change to this scaling factor will persist until
changed again.
229
End Row
SCP/ 123
End Row SCP/123 allows the operator to terminate chemistry on a single row of oligos
without ending the entire run. The Miscellaneous (MISC) field is used to specify which
row is to be terminated: 1 for synthesis, 2 for cleavage, and 3 for purification
(SCP/123). This function operates only in Manual Control so a run must be Interrupted
before it can be used. Upon resumption of the run, any cycle containing a terminated
row will end immediately.
Some care must be exercised when attempting to end a row during a Pause After
interrupt and, in general, it is preferable to avoid doing this altogether. At this point,
synthesis and cleavage both contain the same row to be ended but for the End Row to
take effect, the row would have to be ended in cleavage. This is because the synthesis
row data has been copied over to the cleavage cycle but, in order to fully implement
run extension, the synthesis cycle is not updated with the information on its new row
until the run is resumed.
As an example, if a run is paused after synthesizing row 3, ending the row in
purification would terminate row 2 while ending the row in cleavage would cancel row
3. Attempting at this point to end row 3 in synthesis would not be successful because
control of the un-ended row has already been handed over to cleavage. In this
scenario, to end row 4 without beginning chemistry on it, the run must be resumed
B-8 ABI 3948 System Special Functions
and an operator Interrupt set while the jaws are closing and pressure testing. Once
this Interrupt has taken effect, row 4 may be ended using End Row SCP/123. So while
it is tricky to correctly end a row at a Pause After, the Pause After feature may be used
to enable the operator to end a row after the first step of the next cycle (jaw close).
There are several scenarios where End Row SCP/123 could prove useful. If, for
example, synthesis falters on the final row because of an amidite shortage then the
row can be terminated while the oligos in cleavage and purification continue on to
conclusion. Or a row with a leaky OneStep™ column that appears in the middle of a
run can be skipped over while the other oligos, both before and after that row, are run
to completion.
230
Go Sub On
Fail
Go Sub On Fail provides a means to interject a clean-up or recovery process into a
cycle at the point where a critical delivery has failed. This function recognizes that a
sensor delivery pause is pending and conditionally executes the subroutine number
specified in the Miscellaneous field. (For more on subroutines, see function 287, Go
Sub, et al.)
One key aspect of using this function is that the Go Sub On Fail step and all
subsequent steps in the recovery subroutine must be flagged as Unsafe so that the
run is not paused prior to the recovery being completed.
With any sensor delivery failure, the run resumes at the step that failed, regardless of
the number of steps between the failure and the time the pause takes effect. Possible
uses of a Go Sub On Fail subroutine include preparing a row for a graceful
cancellation (see function 229, End Row SCP/123) or properly resetting conditions for
a successful retry of the failed delivery.
232
Syn Jaw Close
The discussion below the table applies to all the functions listed in the table
232
Syn Jaw Close
234
Clv Jaw Close
236
Pur Jaw Close
325
Jaw Test Times
Auto-resume
The Jaw Close functions listed above can be used without either a Time or a
Miscellaneous (MISC) parameter to simply close the jaws. They may also use these
parameters to optionally invoke a pressure test of the jaw seal when the jaw closes. In
this case, the pressure drop test time is defined by the TIME field. The maximum
allowable pressure drop is declared in the Miscellaneous field in 1/1000's of a psi.
If the “Pause On Jaw Leak” check box in the Instrument Preferences Window is
checked and the jaw test fails, the run will be paused at that point. This may be the
preferred option when the instrument is running with an operator in attendance. If the
“End Row On Jaw Leak” check box in the Instrument Preferences Window is checked
and the jaw test fails, all columns in the module associated with the failed jaw will be
deactivated immediately (see function 229, End Row SCP/123). For throughput
reasons, this is a useful option for an unattended instrument.
To perform a jaw/block pressure test, declare a non-zero pass/fail value for the
pressure drop in the Miscellaneous field. If no pass/fail value is declared in the step,
the default specified by the “Leak OK in 0.01 PSI” setup variable in the Instrument
Preferences Window will be substituted. If the step and default values are both zero,
ABI 3948 System Special Functions B-9
no jaw/block pressure test will be performed. The “Man Cont Jaw Testing” check box in
the Instrument Preferences window must be checked to enable jaw/block pressure
testing based on default values when closing the jaws in manual control. Jaw testing
can also be initiated in manual control by specifying the test parameters when
executing the jaw close.
Jaw Test Times uses the TIME field to define the default pressure drop time for any
jaw/block pressure test. The initial pressurization time in whole seconds (settling time)
is defined in the Miscellaneous field. If this function is not used to define these times, a
default of 30 seconds will be used for both. The times established by this function must
be reinstated after a power failure or database reset.
The Instrument Preferences window contains the testing pressure in psi for jaw/block
pressure tests performed within cycles and defines the terms under which the
auto-resume feature will be enabled. The term “auto-resume” refers to the 3948's
ability to wait for a period and then restart itself after being paused by a sensor
delivery failure. The auto-resume feature is closely tied to the results of the jaw/block
pressure tests and so it is discussed here.
For auto-resume to be enabled, three basic requirements must be met. The first
requirement is for the operator to select the correct settings in the Instrument
Preferences Window. The default settings accomplish this. Secondly, the system must
be shown to be currently leak tight so that auto-resuming will not result in magnifying
spillage due to a leak. Depending on the results of each individual jaw/block leak test,
some active cycles may not be allowed to auto-resume at the same time that others
would be. Finally, auto-resume is an automatic process designed for unattended
operation: control is always surrendered to the operator if there is any manual
intervention with the instrument while an auto-resume is pending.
To meet all three conditions, a number of factors must be in place:
♦
“Pause On Sensor Fail” must be checked in the Instrument Preferences Window
so that sensor delivery pausing is enabled.
♦
“Auto-resume Minutes” in the Instrument Preferences Window must be set to
some value greater than zero (15 minutes is the default).
♦
Jaw/block pressure testing must be in effect and the testing pressure must be
done at the system maximum 12 psi as specified by the “Jaw Leak Test in PSI”
setup variable in the Instrument Preferences Window. A value of 12 psi is the
default for this setup variable.
♦
Jaw/block pressure testing must be performed with a minimum pressure drop time
of 30 seconds. This is the default time provided for by the system but care must be
used if this time is modified either within the jaw close step or by setting an
alternate default using Jaw Test Times.
♦
Jaw/block testing must past the “Auto-res OK 0.01 PSI” test standard in the
Instrument Preferences Window. This allows a maximum pressure drop of only
1.80 psi to pass compared to a maximum allowable drop of 5 psi for the “Leak OK
in 0.01 PSI” preference value.
These setup variable default values are 1.30 and 2.00 psi, respectively (entered in
1/100's p.s.i. as 130 and 200). It is possible for the instrument to “pass” the
jaw/block leak test and run chemistry while not passing a more stringent standard
required to enable auto-resume.
♦
The instrument must be running automatically. Any intervention by an operator
during an auto-resume wait period will cancel the auto-resume. Such operator
B-10 ABI 3948 System Special Functions
interventions include initiating a manual control action, manually resuming the
run, or even “interrupting” an instrument that is paused with an auto-resume
pending. Also, auto-resume will never go into effect while a procedure or other
operation is underway in manual control. However, once a run is resumed (by the
operator), auto-resuming is re-enabled and will go into effect if there are any
future sensor delivery failures within the cycle.
If the above conditions are met, most sensor delivery functions will auto-resume when
they fail to deliver. The exceptions to this are function number 1, B+TET to Syns, and
ramping functions (see the preceding discussion of function 135, Crude A to UV, et al).
251
254
Relay 1 Pulse
and
Relay 2 Pulse
Different external devices, such as fraction collectors, may require different length
pulses to be controlled properly. The pulse width for these functions is determined by
the time entered for the step. Additionally, relay 2 is turned on when a run is
Interrupted, is turned off when the run is Resumed, and is pulsed for two-tenths of a
second when a run ends. This is done to allow relay 2 to trigger an alarm or other
device to alert the operator when a run has paused or ended.
260
Set Coil Temp
This function sets the deprotection heater to the target temperature specified in the
Miscellaneous (MISC) field (in degrees C). If the Miscellaneous field is zero, this
function will substitute the default value specified by the “Xfer Into Coil” setup variable
in the Instrument Preferences Window.
261
Wait Coil
Temp
This function sets the deprotection heater to the target temperature specified in the
Miscellaneous (MISC) field (in degrees C). It then waits the allotted Time for the
specified temperature to be reached. Like a sensor function, if the temperature is
reached before time runs out, the function ends early. If the step runs out of time and
the temperature is further than 2 °C from its target, a message is issued. If the cycle
step calls for either a zero temperature or a zero Time (or both), this function will
substitute the appropriate default value(s) specified in the Instrument Preferences
window (the “Coil Cool Secs” and “Xfer From Coil” setup variables.)
262
Tggle Vlves On
and
Tggle Vlves
Off
Toggling on a valve is like turning on a light in a room. No matter what else happens in
the room, the light remains on until it is turned off. Tggle Vlves On turns on the valve
specified in the Miscellaneous (MISC) field so that it stays on throughout the execution
of other functions. Tggle Vlves Off is used to turn the valve off again. A set of valves
must be toggled on and off one at a time. This feature is used while draining TFA
during purification. At that time, Valve 70 is then toggled so that it temporarily redirects
vessel drains into the halogenated waste bottle.
Set Rmp Fxn
Sns
The discussion below the table applies to all the functions listed in the table.
263
265
265
Set Rmp Fxn Sns
266
Set Rmp Fxn Trp
267
Set Rmp Fxn Chk
268
Set Rmp Fxn Dly
The Set Rmp Fxn functions listed above are used to set parameters that help to define
the behavior of the ramping functions described previously (see function 135,Crude to
UV, et al.). These parameters must be set in each chemistry controller before ramping
functions can be successfully used by that controller. There are four chemistry
ABI 3948 System Special Functions B-11
controllers: synthesis, cleavage, purification, and manual control. The parameter
values may be set differently for each controller. The parameter values are declared in
the Miscellaneous (MISC) field of a Set Rmp Fxn function and set by executing the
function within a given chemistry controller (i.e., within a cycle). Once set, these
parameters will persist until changed.
When activated, sensors continuously sample for the presence of wetness every 12
milliseconds and record a series of “wet” and “dry” readings. Each sensor decides
whether the flow path is wet or dry based on some number of the most recent
readings taken and the proportion of those readings that is wet or dry. Except for
ramping functions, these standards for wet/dry are fixed. The Set Rmp Fxn's allow the
cycle developer to vary these values for ramping functions.
Set Rmp Fxn Sns defines how large a sampling of most recent readings will be
evaluated to decide if the sensor flow path is wet or dry. This number cannot be larger
than 10, the value that is assigned to most non-ramping functions.
Set Rmp Fxn Trp defines how many of the sample readings must be “wet” to
trigger—or trip—the sensor. When dealing with cool, non-gaseous liquids it is
reasonable to set this value high relative to the number of samples assessed (e.g., 9
“wets” out of a set of 10 readings). When dealing with hot, frothy ammonia coming out
of the deprotection coils, a lower standard such as 5 “wets” out of 10 readings is
reasonable.
The “Sns” and “Trp” values are built into all sensor functions but the ramping functions
additionally verify a liquid slug once detected. Set Rmp Fxn Chk defines the number of
“wets” required to verify—or check—the initial trigger and end function execution if
verified. For a 9 out of 10 wet trigger, a 7 out of 10 verify is reasonable and a 5 out of
10 verify is fine for hot ammonia.
Set Rmp Fxn Dly defines the number of milliseconds to pause (delay) after the initial
trigger before beginning to collect readings for the verification. To avoid losing too
much sample past the sensor during verification, this delay should not be too long—50
milliseconds is typical.
If a ramping function does not verify within two and one-half seconds, it resets the
sensor to look again for a new trip followed by a verify. Verification is especially useful
during critical transfers because it allows the function to overlook any small slugs that
might precede the principal slug. Without verification, the actual sample could be lost
whenever a leading slug tricked a transfer into stopping too early.
It is technically possible for a ramping function to trip many times before verifying. In
practice, a second trip is infrequent and a third trip has never been observed.
B-12 ABI 3948 System Special Functions
269
270
Send Message
and
Comment
Send Message and Comment both send a message to be displayed in the instrument
status window on the Mac. Which message to send is defined by the Miscellaneous
(MISC) field. Messages contain instructions for the operator and comments contain
informative descriptions. They are used in maintenance and test procedures such as
bottle changes, sensor calibrations, and leak tests. Available messages and
comments are listed below – some are reserved for possible future needs:
Messages List (for Send Message function 269):
“Place new 2g. amidites on synthesizer.”
// Msg. 0
"Select 'resume' to continue.”
// Msg. 1
“Auto-dilute procedure complete.”
// Msg. 2
“Replace Amidites, Then Select Resume.”
// Msg. 3
“Place empty columns in table pos. 1-9.”
// Msg. 4
“
// Msg. 5
”
“Empty all bottles except Acetonitrile.”
// Msg. 6
“Place empty columns in table pos. 1-3.”
// Msg. 7
“Place empty columns in table pos. 4-6.”
// Msg. 8
“Place empty columns in table pos. 7-9.”
// Msg. 9
“Change reagent bottle now.”
// Msg. 10
“Verify the needle is down in well 1.”
// Msg. 11
“Verify the needle is down in well 8.”
// Msg. 12
“Verify the needle is down in well 40.”
// Msg. 13
“Verify the needle is down in well 48.”
// Msg. 14
“Verify the needle is at left waste.”
// Msg. 15
“Verify the needle is at right waste.”
// Msg. 16
“Verify that all regs display 5 psi.”
// Msg. 17
“System Message 18.”
// Msg. 18
“System Message 19.”
// Msg. 19
Comments List (or Comment function 270):
“ACN (Reg 4)”
“Tet & Bases (Reg 9)”
“NMI, AC2O & I2 (Reg 8)”
“TCA + Acetic Acid (Reg 2)”
“Syn Col A (Reg 7)”
// comment 0
“Syn Col B (Reg 7)”
“Syn Col C (Reg 7)”
// comment 5
ABI 3948 System Special Functions B-13
“Syn VB's (Reg 7)”
“Clv VB's & Bot Depro (Reg 10)”
“Clv Cols (Reg 10)”
271
272
273
274
“Depro Coils (Reg 1)”
“Depro Coil B (Reg 1)”
“Depro Coil C (Reg 1)”
“NH4OH + TEAA (Reg 5)”
“Pur VB's & Up Depro (Reg3)”
// comment 10
“Pur Col A (Reg 3)”
“Pur Col B (Reg 3)”
“Pur Col C (Reg 3)”
“TEAA (Reg 5)”
“TFA + 20% Acn (Reg 6)”
// comment 15
“DI (Reg 5)”
“20% ACN (Reg 6)”
“UV (Reg 3)”
“Vent Amidite v76 (Reg 9)”
“Vent NH4OH v74 (Reg 5)”
// comment 20
“Test(s) completed.”
“Message #26”
“Message #27”
“Message #28”
“Message #29”
// comment 25
Begin of Cycle
and
End of Cycle
These functions serve only to mark the beginning and ending steps of a cycle or
procedure.
Begin Loop
and
End Loop
These functions mark the beginning and ending boundaries of a block of steps that
will be executed repeatedly. The number of times that the steps will be repeated, the
loop counter, is defined in the Miscellaneous (MISC) field of the Begin Loop step.
The block of steps within a loop will always be executed at least once, even if the
Begin Loop parameter is zero. This is because it is the End Loop function at the end of
the block that decides whether the steps have been repeated enough times to drop
out of the loop or not. If not, the loop counter is decrements by 1 and the loop is
repeated, starting with the first step after Begin Loop. When all loops are done, the
cycle continues with the step after the End Loop step. Cycles must always have a
matching number of Begin and End Loop steps.
B-14 ABI 3948 System Special Functions
Loops may be nested, that is, one or more loops may appear inside another loop.
Loops may be nested up to ten levels deep. An example of loop nesting to two levels is
outlined here:
Begin Loop
3
outer loop
5
inner loop
[Steps to execute 3 times]
Begin Loop
[Steps to execute 15 times]
note: 15 = 3 x 5
End Loop
end inner loop
End Loop
end outer loop
When loops are nested, it is the loop countdown counter for the outermost loop that is
displayed in the Chemistry Monitor window. For the above example, the counter
displayed would be the one declared by the first Begin Loop step which would count
down from the initial value of 3.
275
Start Here
This function marks the initial cycle step for the first synthesis cycle only. Normally, a
synthesis cycle begins with the coupling of a new base to the oligo, proceeds through
capping and oxidation, and then detritylates in preparation for another base addition at
the start of the next cycle. In the first synthesis cycle, the 3' base is already attached to
the support so the appropriate point to begin this cycle is with the initial detritylation.
276
Exit DMT On
This function executes only on the last synthesis cycle of any given column and it only
affects sequences that are being synthesized “trityl on” (i.e., purified oligos or “trityl on”
crudes). Its purpose is to prevent oligos from being detritylated on their final synthesis
cycle when no further base additions are forthcoming.
The effect of this function is to deactivate the column(s) that are just concluding
synthesis in time to avoid a final detritylation. Further, continuing synthesis on
columns making longer oligos will have no effect on those columns that have already
finished. Oligos being synthesized “trityl off” have their columns deactivated at Cycle
End.
ABI 3948 System Special Functions B-15
277
If Pur Col A
The discussion below the table applies to all the functions listed in the table.
277
If Pur Col A
280
Else
281
EndIf
291
If Any Act Cols
297
If Crude Col A
to
279
If Pur Col C
to
299
If Crude Col C
The functions listed above are all used to build a programming construct generally
referred to as an if statement. The basic purpose of an if statement is to execute a
series of steps only if a certain condition is true.
The minimal form of an if statement is:
If
steps to do if true
EndIf
continue on.
A more complex variation is:
If
steps to do if true
Else
steps to do if false
EndIf
continue on.
If statements may be nested to any number of levels (see function 273, Begin Loop)
but only one End If is needed with many If's:
If
steps to do if true
If
steps to do if also true
EndIf
continue on.
If the test condition is not met (is false) then the cycle skips the steps between the If
function and the Else or EndIf function, whichever occurs first, and continues
executing from that point. If the test condition is true, then the steps immediately after
the If function are executed and any steps between the Else step (if there is one) and
the EndIf are skipped. The Else and EndIf functions act as markers in the cycle to tell
the If functions what steps to skip.
The If functions in this particular group all involve the status of column activity. Most
likely, these functions would be used only in purification cycles. The If Pur Col
functions can be used to select certain cycle steps for execution only if the oligo for a
given column is to be purified. Similarly, the If Crude Col functions can be used to
select certain cycle steps for execution only if the column is handling a crude oligo.
Instead of operating based on the status of just one column, If Any Act Cols provides a
means to conditionally execute steps for a group of columns involved with purifying
B-16 ABI 3948 System Special Functions
oligos. After using function 282, Select Pur Cols, only those columns that remain
active will be affected by the steps between If Any Act Cols and EndIf.
282
Select Pur
Cols
The discussion below the table applies to all the functions listed in the table.
282
Select Pur Cols
283
Select Act Cols
300
Engage All Cols
319
Revive Act Cols
The Select /Engage/Revive functions are all designed to change the status of which
columns are active and which are not. Normally a column is active if it is processing
an oligo, either crude or purified, and inactive if it is not. Column active status is
initialized automatically at the beginning of each cycle for the set of oligos being
processed during that cycle.
Select Pur Cols will deactivate any columns that are processing crude oligos and
leave only those columns active that are processing purified oligos. Using this function
in conjunction with If Any Act Cols provides a convenient means to restrict purification
activities to just those columns that need them.
Engage All Cols ensures that all columns are active, even if they are not all currently
processing oligos. One use for this feature is to guarantee that all of the coils are
cleaned as needed. Otherwise, if a single oligo is being cleaved by the current cycle,
only the coil for that oligo would get cleaned—even if the previous cycle had “dirtied”
all three coils by filling them with oligos to be deprotected.
Revive Act Cols will restore to active status any column that was set up for synthesis
but that has since been deactivated. This mechanism may be used to reactivate
columns that were turned off because their syntheses had been concluded previously.
When used in conjunction with If Cyc Greater, this function can enable the execution
of the “end procedure” on all synthesizing columns during the last synthesis of the
longest oligo in a set of three. This is useful for creating cycles to manage special
additions at the 5´ end, i.e., extra washing after dye labelling or extra detritylation for
certain modified aminos.
Revive Act Cols may also be used to reactivate columns that have been ended due to
a jaw/block leak test failure or by the use of the End Row SCP/123 (see function
number 229 above). By using this function in the purification cycle, oligos in the coils
may be recovered even though the jaw/block leak test has failed.
Select Act Cols undoes the effects of the other three related functions by properly
reselecting only normally active columns, i.e. just those columns actually processing
oligos, regardless of type. If Select Pur Cols has turned off any columns processing
crude oligos, this function will reactivate those columns. Similarly, columns will be
deactivated if they are columns that have been reactivated by Revive Act Cols or
columns without oligos that have been activated by Engage All Cols.
ABI 3948 System Special Functions B-17
284
Clv Wants
Depro
The discussion below the table applies to all the functions listed in the table.
284
Clv Wants Depro
285
Pur Owns Depro
286
Clv Owns Depro
These three functions are used by cleavage and purification cycles to arbitrate
between the two systems regarding the shared ownership of the deprotection coils.
Pur Owns Depro is executed early in the purification cycle to declare that the
purification system has control of the coils. Once the deprotected oligos are
transferred out of the coils into the purification vessels, the purification cycle executes
Clv Owns Depro to surrender control of the coils to the cleavage system.
The cleavage cycle is responsible for cleaning out any residue from previously
deprotected oligos before transferring in the most recently cleaved oligos. A Clv Wants
Depro step is placed in the cleavage cycle immediately before this cleaning begins.
The cleavage cycle waits at this step as long as the purification system retains
ownership of the coils. Control of the coils is eventually transferred to cleavage (Clv
Owns Depro) after the purification cycle has removed the previous set of deprotected
oligos from the coils. At this point, the cleavage cycle moves on to clean the coils,
transfer in the current set of oligos, and start deprotection.
Since there is no purification cycle running during the first cleavage cycle, Clv Owns
Depro is executed in the begin procedure to ensure that the cleavage system is in
possession of the coils when the first set of oligos in a run is ready for deprotection.
287
Go Sub
The discussion below the table applies to all the functions listed in the table.
287
Go Sub
288
Return Sub
289
Sub Label
290
Goto End
The set of functions above is involved with subroutines. A subroutine is a set of steps
that accomplishes a particular task, such as filling or draining a set of vessels,
performing a detritylation, or cleaning a transfer path. A cycle or procedure may have
many subroutines.
Subroutines are always placed at the end of the cycle, after a Goto End step. The
Goto End step is a marker that says to ignore all of the steps in the subroutines that
follow and jump directly to the Cycle End step. Effectively, the Goto End step is the last
step of the main cycle.
The Sub Label function marks the beginning of a subroutine. The Miscellaneous
(MISC) field in the Sub Label step uniquely identifies a specific subroutine. All of the
subroutines within a given cycle or procedure must have different numbers.
Subroutines are only recognized within their cycle so the same Sub Label may be
applied to different subroutines within different cycles.
By using Go Sub, a subroutine may be “called” from anywhere in the main cycle to do
its job. Calling a subroutine means that cycle execution jumps from the main cycle
down to the first step of the subroutine and begins executing the steps following the
B-18 ABI 3948 System Special Functions
Sub Label. The Miscellaneous field in a Go Sub step identifies the corresponding Sub
Label step to jump to. Subroutines may not be called from other subroutines.
Return Sub marks the end of a subroutine. The steps in between the Sub Label and
Return Sub steps constitute the working body of the subroutine. When a Return Sub
step is reached, execution jumps back up to the main cycle and resumes at the step
following the Go Sub step that called the subroutine. Column activity status may be
adjusted by Return Sub (see Go Sub A/B/C below).
Subroutines are useful when the work of the subroutine must be done a number of
times in different places in a cycle. With subroutines, only one copy of the steps
required to do a given job needs to be created. Cycles may be written with fewer steps
because every time the same steps recur in a cycle they can be replaced by a single
Go Sub call. Also, if a subroutine's process ever needs alteration, changes need only
be made in just one place instead of in several locations.
291
If Any Act
Cols
See preceding discussion of function 277, If Pur Col A, et al.
292
Go Sub A
to
Go Sub C
These functions, called conditional Go Sub's, are a variation of the standard Go Sub
function with a Select function twist (see function 282, Select Pur Cols). Each of these
functions executes a subroutine call to the Sub Label step with a matching
Miscellaneous (MISC) field value. However, this set of Go Sub's will execute the
subroutine call only if their corresponding column is active.
294
A second feature of conditional Go Sub's is that when the subroutine is called, any
other active columns are deactivated for the duration of the subroutine's execution.
When a conditional Go Sub call is used, the Return Sub function automatically
restores column activity status to what it was when the subroutine call was made. This
status may be all columns active, only purification columns active, etc. (see function
282, Select Pur Cols, et al.)
Conditional Go Sub's take advantage of the fact that almost all column delivery
functions come in sets consisting of one parallel version of the function and three
serial, column-specific versions (e.g., ACN to Syn Cols and ACN to Syn Col A, B, or
C). When only one column is active, the parallel version of a function will behave
exactly like the serial version for that active column. If a subroutine is written using
parallel functions and then called conditionally, it will act like a subroutine written for
one specific column.
Conditional subroutine calls may be used to help minimize the total number of cycle
steps, simplify cycle updates, and maximize the uniformity of cycle execution across
columns.
ABI 3948 System Special Functions B-19
295
If First Cycle
The discussion below the table applies to all the functions listed in the table.
295
If First Cycle
296
If Last Cycle
316
If Cyc Greater
This is another category of If functions which must be paired with an Endif as
discussed above (see function 277, If Pur Col A). These functions are only valid in a
synthesis cycle. They cause a block of steps to be conditionally executed or skipped
over based on the number of the cycle currently executing.
The block of steps between If First Cycle and its corresponding Else or Endif step will
only be executed if the current cycle in progress is the first cycle of synthesis.
Similarly, the block of steps between If Last Cycle and its corresponding Else or Endif
step will only be executed if the current cycle in progress is the last cycle of the longest
oligo being synthesized in a given row. These functions are useful for creating “begin -”
and “end procedures” for synthesis cycles which may be used for instrument
housekeeping purposes or to implement special cycle variations. When implementing
special 5´ monomer cycles, If Last Cycle will usually be used in conjunction with
Revive Act Cols (see the preceding discussion of function 282, Select Pur Cols, et al).
The If Cyc Greater function enables the synthesis cycle to modify or adapt its activities
as the oligo it creates gets longer. The Miscellaneous (MISC) field is used to define
the cycle number at which synthesis will begin to execute additional steps. By nesting
If Cyc Greater steps (see function 277, If Pur Col A), new activities may be gradually
incorporated into the cycle as the oligo reaches new milestone lengths.
Having an adaptive cycle removes the requirement to have multiple cycles for different
length oligos. It also allows the chemistry to perform optimally at each oligo length
instead of attempting to strike a balance between the activity required during early
cycles and that required later on. This capability facilitates minimizing cycle time and
maximizing cycle performance.
297
299
If Crude Col A See preceding discussion of function 277, If Pur Col A, et al.
to
If Crude Col C
300
Engage All
Cols
316
If Cyc Greater See the preceding discussion of function 295, If First Cycle, et al.
317
Save Reg Pres
and
Restore
RegPres
Both of these functions use the Miscellaneous (MISC) field to declare the number of
the regulator involved. Save Reg Pres will save the regulator pressure setting value of
the specified regulator. Restore RegPres resets the pressure setpoint of the regulator
to the value saved by Save Reg Pres. Regulator pressures may be saved and restored
by subroutines that alter the regulator's pressure during their execution.
Revive Act
Cols
See the preceding discussion of function 282, Select Pur Cols, et al.
318
319
See the preceding discussion of function 282, Select Pur Cols, et al.
B-20 ABI 3948 System Special Functions
320
Check Snsr
Cal
This function checks that the wet sensor calibration values are at least twice the dry
values for all sensors and issues a Critical Message if any sensor calibration fails to
meet this standard. Check Snsr Cal is used in the standard begin procedure and in the
sensor calibration procedure(s).
325 Jaw Test Times See the preceding discussion of function 232, Syn Jaw Close, et al.
329 Begin Leak Test The Leak Test functions mark the begin and end of a pressure/leak test. A Wait step is
placed between these two functions to define the pressure drop time for the test.
and
330 End Leak Test
The Time field in a Begin Leak Test step defines how much time to use taking an initial
baseline pressure average. The Miscellaneous (MISC) field defines which regulator
pressure is being tested.
The Time field in an End Leak Test step defines how much time to use taking a final
average pressure reading. The Miscellaneous field defines the pass/fail pressure drop
threshold for the test in 1/100's of a psi.
When the pass/fail threshold is less than 1 psi, a leak test is being performed and the
test passes when the pressure drop from initial to final reading is less than the
threshold. When the pass/fail threshold is greater than 1 psi, a vent test is being
performed and the test passes when the pressure drop is greater than the threshold.
396
Time Stamp
This function enters the ABI 3948 System’s current time-of-day into the Microphone
log. This time stamp is tagged with the number in the Miscellaneous (MISC) field. The
convention is that time stamps for cleavage are tagged with the numbers 20 through
29 and the time stamps for purification are tagged 30 through 39.
Time stamps were created as an aid to cycle development so that timing information
for the various parts of any cycle might be easily tracked. When a time stamp is
logged, the time of the previous time stamp for the given chemistry controller is also
reported. This makes it easier to calculate the duration between the time stamps
bracketing the start and end of some portion of a cycle.
401 SynUpr Wet 401 The last 100 functions on the ABI 3948 System function list are user functions that can
perform sensor-controlled deliveries. Like standard user functions, the valve list for
to
these functions can be defined by the operator. These functions offer the additional
500 UV Dry 500
advantage of having sensors pre-assigned to them. These sensors are engaged in the
usual fashion by setting the SNS field in the step to YES. The particular sensor or set
of sensors tied to a sensor user function is specified in the default function name. As
with standard user functions, the function name may be edited as well as the valve list.
There are three components to the default names provided for sensor user functions.
The first part of the default name identifies the sensor or sensors associated with the
function. The second component spells out whether the function is “wet” or “dry”, that
is, whether the function's sensor(s) trip upon detecting wetness (liquid delivery) or
dryness (gas delivery, flush or back flush). In keeping with the convention for standard
user functions, the function number is included as the final component of the default
function name.
ABI 3948 System Special Functions B-21
As with other sensor functions, parallel sensor user functions are always grouped on
the function list together with their associated serial functions. So SynUpr Wet 401 is
followed by SynUprWet A 402, SynUprWet B 403, and SynUprWet C 404.
Note
Parallel sensor functions do not have valve lists of their own but use the valve lists of
the associated serial sensor functions. Valve assignments must be made properly for all serial
functions of a given type for the parallel sensor function to work or work properly. The
SynUpr, SynMan, and SynLow refer respectively to the upper synthesis sensors
numbered 1 to 3, the synthesis manifold sensors numbered 22- to 24, and the lower
synthesis sensors numbered 4 to 6. Sensors 7 to 9 are the Clv sensors, sensors 10 to
12 are the Coil sensors, and sensors 14 to 16 are the Pur sensors. Sensor 21 is the
UV sensor. (See the Plumbing Diagram in Appendix E to verify which sensors are
associated with which columns. In some instances, the column alphabetic order and
sensor numeric order are reversed.)
As with other sensor functions, parallel sensor user functions are always grouped on
the function list together with their associated serial functions. So SynUpr Wet 401 is
followed by SynUprWet A 402, SynUprWet B 403, and SynUprWet C 404.
This grouping of sensor functions into sets is crucial to the operation of parallel
functions. Parallel functions do not use their own valve lists to execute, they use
the valve lists of their associated serial functions instead. In this way, parallel
functions will turn on only the valves for active columns by merging together the valve
list for each active column's associated serial function and excluding the valve lists of
functions associated with inactive columns. When one column finishes its delivery
before the others, its single set of valves is deactivated. Any other active columns'
valves will remain on until their sensors trigger.
User definable functions operate according to the same scheme. In order to program
the valve list for a parallel user function, the appropriate valve lists for each associated
serial function must be entered. The value to also entering the complete, three-column
valve set into the parallel function's valve list is one of documentation: the entire valve
list may then be viewed under one function in Manual Control.
B-22 ABI 3948 System Special Functions
Cycles, Procedures, and
System Messages
C
C
In This Appendix
Introduction This appendix contains listings of the standard synthesis, cleavage/deprotection, and
purification cycles for use in understanding ABI 3948 System chemistry.
Topics Covered This appendix contains the following topics:
Topic
See page
ABI 3948 System Chemistry
C-2
Overview
C-2
The Standard Synthesis Cycle
C-2
Synthesis Cycle Summary
C-2
Synthesis Cycle Listing
C-4
The Standard Cleavage/Deprotection Cycle
C-8
Cleavage/Deprotection Cycle Summary
C-8
Cleavage/Deprotection Cycle Listing
C-11
The Standard Purification Cycle
C-17
Purification Cycle Summary
C-17
Purification Cycle Listing
C-22
Procedures
C-31
General
C-31
Edit Begin Procedure View
C-31
Edit End Procedure View
C-33
Edit Bottle Procedure View
C-35
Miscellaneous Procedures
C-36
System Messages
C-37
Instrument Status and Message Indicators
C-37
Types of Messages/Categories of Messages/Conventions
C-37
Regular Reports Messages
C-38
Critical Messages
C-40
Microphone-only Messages
C-40
Hierarchical Messages
C-41
Cycles, Procedures, and System Messages C-1
3948 Chemistry
Overview Chemistry Protocols
Chemistry in the ABI 3948 System is performed by assigning a protocol to each
oligonucleotide to be produced. A protocol consists of three cycles to perform the
three types of chemistry: synthesis, cleavage/deprotection, and purification. Each
cycle is a series of functions which operate the valves and perform the other actions
needed to produce an oligonucleotide.
Functions in Cycles
Functions are arranged in a particular order to create a cycle, which performs an
operation. One Synthesis cycle, for example, contains all the functions necessary for
one base addition. The functions in each standard cycle have optimized delivery times
to ensure completion of each chemical reaction with minimal reagent consumption
and minimal unwanted side reactions.
User Cycles
Besides the standard cycles, you can develop up to 34 additional cycles, 18 for
synthesis, 8 for Cleavage/Deprotection, and 8 for Purification. Although the standard
cycles cannot be changed or deleted, they can be copied into one of the user cycle
locations and edited with the Cycle Editor to provide the basis for a new user cycle.
Synthesis Scale
The ABI 3948 System synthesizes at one scale only. Synthesis of a 25-mer typically
yields 8.0-12 ODU of crude oligonucleotide, which when purified may equal 0.5 to 4
ODU.
C-2 Cycles, Procedures, and System Messages
The Standard Synthesis Cycle
Synthesis Cycle The Synthesis cycle processes/reactions needed for synthesis are illustrated in the
Summary following flow chart (Figure C-1) and the step numbers corresponding to these
activities are listed in Table C-1. Table C-2 contains a listing of the standard synthesis
cycle.
Figure C-1 Synthesis Cycle Flow Chart
Cycles, Procedures, and System Messages C-3
Table C-1 Summary of Synthesis Cycle
Step Range
Description
47 - 52
Start Here, Leak Test
53 - 67
68 - 86
87 - 100- 44
1 - 19
20 - 31
32 - 46
C-4 Cycles, Procedures, and System Messages
♦
Close synthesis jaw, begin leak test
and check for pass or fail.
♦
If leak test passes, go to if first cycle.
♦
If leak test fails, go to cycle end.
ACN Wash
♦
If first cycle, give columns extra ACN
wash.
♦
Normal column(s) ACN wash.
♦
Normal column(s) ACN wash
♦
If last cycle, synthesis module cleanup
(Go Sub 3).
Detritylation
♦
Exit DMT On, for “purified or trityl on
crude’s” only.
♦
This function executes only on the last
synthesis cycle for each column.
♦
7 loop of detritylation (Go Sub 3).
♦
If Cyc Greater, additional detritylation
for longer oligos (Go Sub 2).
♦
If first cycle, additional detritylation for
support bound nucleoside (Go Sub 2).
Trityl Wash
♦
Wash and flush to trityl waste with
ACN.
♦
If last cycle, execute synthesis module
cleanup (Go Sub 3) in the case of “trityl
off crudes”.
Begin of Cycle, Base+Tet (Coupling)
♦
Tetrazole and Base+Tet delivery.
♦
Coupling push/wait loop
♦
If Cyc Greater, extend coupling for
longer oligos.
Capping
♦
Deliver capping reagents to manifold
sensors.
♦
Capping push/wait loop
Oxidation
♦
Deliver Iodine to manifold sensors.
♦
Iodine push/wait loop
Synthesis Cycle The annotated listing below is provided to aid you in understanding ABI 3948 System
Listing synthesis:
Table C-2 Standard Synthesis Cycle (v4.20)
STEP
FUNCTION
NUM
TIME
MISC
SNS
SAFE
Comments
1)
Begin of Cycle
271
0.0
0
Yes
Yes
Marks beginning of cycle
2)
SynUprBlk Flush
105
2.0
0
No
Yes
Flush out upper synthesis block
3)
TET to Syn Cols
48
5.0
0
Yes
Yes
Del. Ieading TET slug to lower sensors
4)
B+ TET to Syns
1
0.0
0
Yes
Yes
Push TET to manifold sensors w/Base+Tet
5)
ACN to SynWaste
47
2.0
0
No
Yes
Rinse and prime lower block w/ACN
6)
ACN to Syn Cols
43
15.0
0
Yes
Yes
Push Base+TET to upper sensor w/ACN
7)
Begin Loop
273
0.0
5
No
Yes
Begin coupling push/wait loop
8)
Coupling Wait
26
8.0
0
No
Yes
Wait for Coupling (from B+TET table)
9)
iif after cycle/base 6O,
If Cyc Greater
316
0.0
60
No
Yes
10)
Wait
259
2.0
0
No
Yes
11)
If Cyc Greater
316
0.0
75
No
Yes
12)
Wait
259
2.0
0
No
Yes
13)
If Cyc Greater
316
0.0
90
No
Yes
14)
Wait
259
2.0
0
No
Yes
15)
Endif
281
0.0
0
No
Yes
End of all "if cyc greater"
16)
ACN to SynCol A
44
0.3
999
No
Yes
Push amidite to column A w/ACN or wait
17)
ACN to SynCol B
45
0.3
999
No
Yes
Push amidite to column B w/ACN or wait
18)
ACN to SynCol C
46
0.3
999
No
Yes
Push amidite to column C w/ACN or wait
19)
End Loop
274
0.0
0
No
Yes
End coupling loop
20)
SynLowBlk Flush
65
1.0
0
No
Yes
Flush out lower synthesis block
21)
Go Sub
287
0.0
1
No
Yes
Flush columns/lines/blocks
22)
CAP to Syn Cols
27
15.0
0
Yes
Yes
Deliver CAP to manifold sensors
23)
ACN to SynWaste
47
1.5
0
No
Yes
Rinse and prime lower block w/ACN
24)
ACN to Syn Cols
43
15.0
0
Yes
Yes
Push CAP to upper sensors w/ACN
25)
Wait
259
1.0
0
No
Yes
Initial capping wait, all columns
26)
Begin Loop
273
0.0
5
No
Yes
Begin capping push/wait loop
27)
ACN to SynCol A
44
0.3
999
No
Yes
Push CAP to column A w/ACN or wait
28)
ACN to SynCol B
45
0.3
999
No
Yes
Push CAP to column B w/ACN or wait
29)
ACN to SynCol C
46
0.3
999
No
Yes
Push CAP to column C w/ACN or wait
30)
Wait
259
1.4
0
No
Yes
Wait for capping, all columns
31)
End Loop
274
0.0
0
No
Yes
End capping push/wait loop
32)
ACN to SynWaste
47
1.0
0
No
Yes
Rinse lower block w/ACN
33)
SynLowBlk Flush
65
1.5
0
No
Yes
Flush out lower synthesis block
34)
Go Sub
287
0.0
1
No
Yes
Flush columns/lines/blocks
35)
IODINE to Syns
33
15.0
0
Yes
Yes
Deliver iodine to manifold sensors
36)
ACN to SynWaste
47
1.0
0
No
Yes
Rinse and prime lower block w/ACN
37)
ACN to Syn Cols
43
15.0
0
Yes
Yes
Push iodine to upper sensors w/ACN
38)
Wait
259
1.0
0
No
Yes
Initial oxidation wait, all columns
extend wait for coupling
iif after cycle/base 75,
further extend wait for coupling
iif after cycle/base 9O,
further extend wait for coupling
Cycles, Procedures, and System Messages C-5
Table C-2 Standard Synthesis Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
39)
Begin Loop
FUNCTION
273
0.0
5
No
Yes
Begin oxidation push/wait loop
40)
ACN to SynCol A
44
0.3
999
No
Yes
Push iodine to column A w/ACN or wait
41)
ACN to SynCol B
45
0.3
999
No
Yes
push iodine to column B w/ACN or wait
42)
ACN to SynCol C
46
0.3
999
No
Yes
push iodine to column C w/ACN or wait
43)
Wait
259
1.4
0
No
Yes
Wait for oxidation, all columns
44)
End Loop
274
0.0
0
No
Yes
End oxidation push/wait loop
45)
SynLowBlk Flush
65
1.5
0
No
Yes
Fiush out lower synthesis block
46)
Go Sub
287
0.0
1
No
Yes
Flush columns/lines/blocks .
47)
Start Here
275
0.0
0
No
Yes
First cycle begins here, detritylation
48)
Syn Jaw Close
232
0.0
0
No
Yes
Close syn jaw, start syn module leak test
49)
If Any Act Cols
291
0.0
0
No
Yes
Test for syn module leak test failure
50)
Else
280
0.0
0
No
Yes
No action if passes
51)
Goto End
290
0.0
0
No
Yes
Skip to end of cycle on failed leak test
52)
Endif
281
0.0
0
No
Yes
End of syn module leak test
53)
If First Cycle
295
0.0
0
No
Yes
First cycle only, lines may be wet
54)
ACN to SynWaste
47
5.0
0
No
Yes
Rinse lower block w/ACN
55)
SynLowBlk Flush
65
5.0
0
No
Yes
Flush out lower synthesis block
56)
Flush Syn Col A
54
3.0
0
No
Yes
Flush column A
57)
Flush Syn Col B
55
3.0
0
No
Yes
Flush column B
58)
Flush Syn Col C
56
3.0
0
No
Yes
Flush column C
59)
Go Sub
287
0.0
1
No
Yes
Flush columns/lines/blocks
60)
Go Sub
287
0.0
4
No
Yes
Deliver ACN to upper sensors
61)
Go Sub
287
0.0
1
No
Yes
Flush columns/lines/blocks
62)
Endif
281
0.0
0
No
Yes
End first cycle line clean
63)
Go Sub
287
0.0
4
No
Yes
Deliver ACN to upper sensors
64)
Go Sub
287
0.0
1
No
Yes
Flush columns/lines/blocks
65)
If Last Cycle
296
0.0
0
No
Yes
If last cycle, clean lines/blocks
66)
Go Sub
287
0.0
3
No
Yes
Last cycle wash cola/lines/blocks
67)
Endif
281
0.0
0
No
Yes
End if last cycle line/block clean
68)
Exit DMT On
276
0.0
0
No
Yes
End here if trityl on
69)
SynLowBlk Flush
65
1.0
0
No
Yes
Flush out lower synthesis block
70)
TCA to Syn Cols
38
4.0
0
No
No
Start split delivery of TCAto columns
71)
TCA to Syn Cols
38
15.0
0
Yes
No
Finish split delivery of TCA to sensors
72)
Begin Loop
273
0.0
7
No
No
Begin detritylation loop
73)
Go Sub
287
0.0
2
No
No
Serial TCA (detritylation) push
74)
End Loop
274
0.0
0
No
No
End detritylation loop
75)
If Cyc Greater
316
0.0
60
No
No
if after cycle/base 60, additional TCA push
76)
Go Sub
287
0.0
2
No
No
Serial TCA (detritylation) push
77)
If Cyc Greater
316
0.0
75
No
No
if after cycle/base 75, additional TCA push
78)
Go Sub
287
0.0
2
No
No
Serial TCA (detritylation) push
79)
If Cyc Greater
316
0.0
90
No
No
if after cycle/base 90, additional TCA push
C-6 Cycles, Procedures, and System Messages
Comments
Table C-2 Standard Synthesis Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
80)
Go Sub
FUNCTION
287
0.0
2
No
No
Serial TCA (detritylation) push
Comments
81)
Endif
281
0.0
0
No
No
End of all "if cyc greater tests
82)
If First Cycle
295
0.0
0
No
No
First cycle only, extra detritylation
83)
Begin Loop
273
0.0
3
No
No
Begin first cycle oniv detritylation loop
84)
Go Sub
287
0.0
2
No
No
Serial TCA (detritylation) push
85)
End Loop
274
0.0
0
No
No
End first cycle only detritylation loop
86)
Endif
281
0.0
0
No
No
End "if first cycle"
87)
LowTrityl Flush
106
1.0
0
No
No
Flush lower block to halogenated wate
88)
ACN to Trityl
108
2.0
0
No
No
Rinse lower block to halogenated wate
89)
LowTrityl Flush
106
2.0
0
No
No
Flush lower block to halogenated wate
90)
Waste to Trityl
109
0.0
999
No
No
Redirect v22 waste to v23 halo waste
columns/lines/blocks
91)
Go Sub
287
0.0
1
No
No
Flush columns/lines/blocks .
92)
Go Sub
287
0.0
4
No
No
Deliver ACN to upper sensors
93)
Go Sub
287
0.0
1
No
No
Flush columns/lines/blocks
94)
Flush Syn Col A
54
2.0
0
No
Yes
Flush column A, extra dry step
95)
Flush Syn Col B
55
2.0
0
No
Yes
Flush column B, extra dry step
96)
Flush Syn Col C
56
2.0
0
No
Yes
Flush column C, extra dry step
97)
Waste to Trityl
109
0.0
0
No
Yes
Restore v22 waste output
98)
If Last Cycle
296
0.0
0
No
Yes
If last cycle, wash of cola/lines/blocks
99)
Go Sub
287
0.0
3
No
Yes
Last cycle wash cola/lines/blocks
100)
Endif
281
0.0
0
No
Yes
End if last cycle
101)
Goto End
290
0.0
0
No
Yes
Skip subroutines to end of synthesis cycle
102)
Sub Label
289
0.0
1
No
No
Flush columns/lines/blocks
103)
SynLowBlk Flush
65
2.0
0
No
No
Flush out lower synthesis block
104)
Flush Syn Cols
53
15.0
0
Yes
No
Flush lines thru columns to upper sensors
105)
Flush Syn Cols
53
1.0
0
No
No
Short flush above upper sensors
106)
Return Sub
288
0.0
0
No
No
End of Sub 1
107)
Sub Label
289
0.0
2
No
No
Serial TCA (detritylation push
108)
TCA to SynCol C
41
1.0
999
No
No
Push TCA to column C
109)
TCA to SynCol B
40
1.0
999
No
No
Push TCA to column B
110)
TCA to SynCol A
39
1.0
999
No
No
Push TCA to column A
111)
Return Sub
288
0.0
0
No
No
End of Sub 2
112)
Sub Label
289
0.0
3
No
Yes
Last cycle wash cola/lines/blocks
113)
Revive Act Cols
319
0.0
0
No
Yes
Make all starting columns active again
114)
LowTrityl Flush
106
10.0
0
No
Yes
Flush lower block to halogenated waste
115)
SynLowBlk Flush
65
10.0
0
No
Yes
Flush out lower synthesis block
116)
ACN to Syn Cols
43
2.0
0
No
Yes
Start split delivery of ACN
117)
ACN to Syn Cols
43
15.0
0
Yes
Yes
Finish split delivery of ACN to sensors
118)
Flush Syn Col A
54
20.0
0
No
Yes
Flush colum A to dryness
119)
Flush Syn Col B
55
20.0
0
No
Yes
Flush colum B to dryness
Cycles, Procedures, and System Messages C-7
Table C-2 Standard Synthesis Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
120)
Flush Syn Col C
FUNCTION
56
20.0
0
No
Yes
Flush colum C to dryness
121)
ACN to SynWaste
47
2.0
0
No
Yes
Rinse lower block w/ACN
122)
SynLowBlk Flush
65
10.0
0
No
Yes
Flush out lower synthesis block
123)
Select Act Cols
283
0.0
0
No
Yes
Switch off all previously completed
columns
124)
Return Sub
288
0.0
0
No
Yes
End of Sub3
125)
Sub Label
289
0.0
4
No
No
Deliver ACN to upper sensors
126)
ACN to Syn Cols
43
4.0
0
No
No
Start split delivery of ACN
127)
ACN to Syn Cols
43
15.0
0
Yes
No
Finish split delivery of ACN to sensors
128)
ACN to Syn Cols
43
0.5
0
No
No
Short ACN push past sensors
129)
Return Sub
288
0.0
0
No
No
End of Sub 4
130)
End of Cycle
272
0.0
0
Yes
Yes
Marks end of cycle and all sub routines
C-8 Cycles, Procedures, and System Messages
Comments
The Standard Cleavage/Deprotection Cycle
Cleavage/ The Cleavage/Deprotection cycle processes/reactions needed for oligo production are
Deprotection Cycle illustrated in the following flow chart (Figure C-2) and the step numbers corresponding
Summary to these activities are listed in Table C-3. Table C-4 contains a listing of the standard
cleavage/deprotection cycle:
Figure C-2 Cleavage/Deprotection Cycle Flow Chart
Cycles, Procedures, and System Messages C-9
Table C-3 Summary of Cleavage/Deprotection Cycle
Step Range
Description
1 - 10
Test for Active Columns
11 - 17
18 - 33
34 - 59
60
♦
Test oligos that have failed synthesis
module leak test (failed columns will not
be active.
♦
If synthesis module failed leak test,
then (a) wait for synthesis and
purification modules to complete leak
testing, (b) wait until the deprotection
coils are free and (c) clean up the
cleavage module plumbing (Go Sub 9).
(Go to cycle end).
♦
If columns Active, then close the
synthesis jaw.
Leak Test, Start Here if Columns are Active
♦
Close the cleavage jaw, begin leak test
and check for pass or fail.
♦
If cleavage module failed, the (a) wait
until the deprotection coils are free and
(b) clean up the cleavage module
plumbing (Go Sub 9). (Go to cycle
end).
♦
If cleavage module passed leak test,
then start normal cleavage cycle.
Cleaning of Cleavage Vessels, Prep for
Crude Oligos
♦
Water wash and flush of lower cleavage
block.
♦
Water wash, flush and drying of
cleavage columns, vessels and lines.
Ammonia Cleave of Oligo from the Support
♦
Start Cleavage by delivering ammonia
to the upper cleavage sensors. Rinse
ammonia from the lower cleavage
block. Wait while cleaving.
♦
Set Regulator 10 for the first push of
fresh ammonia over the columns (Go
Sub 1). Wait while cleaving.
♦
Begin cleavage loop, push fresh
ammonia over the columns (Go Sub 1)
and wait while cleaving.
Wait for Purification to Surrender the
Deprotection Coils to Cleavage
♦
C-10 Cycles, Procedures, and System Messages
Cleavage wants the deprotection coils
Table C-3 Summary of Cleavage/Deprotection Cycle (continued)
Step Range
Description
61 - 74
Prepare and Transfer Oligos from the
Cleavage Vessels to the Deprotection Coils
75
♦
Set coil temperature for the transfer
INTO the coils. Wash the transfer line,
coils and cleavage blocks.
♦
Set the ramping function parameters.
♦
Gently consolidate sample into
cleavage vessels.
♦
Ramping transfer into coils from
cleavage vessels.
Start Deprotection Heater
♦
76 - 93
94 - 107
Start the clock for deprotection time
(set in preferences) once the target
temperatures reached.
Column Strip of Uncleaved Product
♦
Start column strip, deliver ammonia to
upper cleavage sensors. Rinse
ammonia from the lower cleavage
block. Wait while stripping.
♦
Set regulator 10 for the first push of
fresh ammonia over the columns (Go
Sub 1). Wait while stripping.
♦
Begin column stripping loop, push fresh
ammonia over the columns (Go Sub 1)
and wait while stripping.
Wash, Drain and Dry Cleavage Module
♦
Drain an dry cleavage module.
♦
Water and ACN washes of columns,
vessels and lines.
♦
Drain an dry cleavage module.
Cycles, Procedures, and System Messages C-11
Cleavage/ The annotated listing below is provided to aid you in understanding ABI 3948 System
Deprotection Cycle cleavage and deprotection:
Listing
Table C-4 Standard Cleavage Cycle (v4.20)
STEP
FUNCTION
NUM
TIME
MISC
SNS
SAFE
Comments
1)
Begin of Cycle
271
0.0
0
No
Yes
Marks beginning of cycle
2)
If Any Act Cols
291
0.0
0
No
Yes
Test for oligos that failed in synthesis
3)
Else
280
0.0
0
No
No
If ok do nothing, else clean civ plumbing
4)
Wait
259
60.0
0
No
Yes
Wait for syn/pur module leak testing
5)
Wait
259
60.0
0
No
No
Wait for syn/pur module leak testing
6)
Wait
259
60.0
0
No
Yes
Wait for syn/pur module leak testing
7)
Clv Wants Depro
284
0.0
0
No
Yes
Wait until deprotection coils are free
8)
Go Sub
287
0.0
9
No
Yes
Drain/wash/dry of coils & depro blocks
9)
Goto End
290
0.0
0
No
Yes
Skip over subroutines to cycle end
10)
Endif
281
0.0
0
No
Yes
To reach this step, columns are active
11)
Clv Jaw Close
234
0.0
0
No
Yes
Close civ jaw, start civ module leak test
12)
If Any Act Cols
291
0.0
0
No
Yes
Test for module leak test failure
13)
Else
280
0.0
0
No
Yes
No action if test passes
14)
Clv Wants Depro
284
0.0
0
No
Yes
Leak test failed, wait for depro coils
15)
Go Sub
287
0.0
9
No
Yes
Drain/wash/dry of coils & blocks
16)
Goto End
290
0.0
0
No
Yes
Skip over subroutines to cycle end
17)
Endif
281
0.0
0
No
Yes
To reach this step, the leak test passed
18)
Time Stamp
396
0.0
20
No
Yes
Record current time
19)
Set Pres Reg 10
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
20)
Clv Blk Flush
144
5.0
0
No
Yes
Flush lower cleavage blocks, may be wet
21)
H2O to ClvWaste
130
5.0
0
No
Yes
Wash lower cleavage blocks
22)
Clv Blk Flush
144
5.0
0
No
Yes
Flush lower cleavane blocks
23)
Begin Loop
273
0.0
2
No
Yes
Begin vessel/column/line water wash
24)
Gas to Clv Cols
121
8.0
0
No
Yes
Dry vessels/columns/lines
25)
Gas to ClvCol A
122
3.0
0
No
Yes
Dry vessel/column/line A
26)
Gas to ClvCol B
123
3.0
0
No
Yes
Drv vessel/column/line B
27)
Gas to ClvCol C
124
3.0
0
No
Yes
Dry vessel/column/line C
28)
Go Sub
287
0.0
7
No
Yes
Delivery of water to sensor
29)
Go Sub
287
0.0
6
No
Yes
Serial 5 psi gas bubble of vessel
30)
Set Pres Reg 10
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
31)
Go Sub
287
0.0
4
No
Yes
Drain vessel/column/line
32)
Go Sub
287
0.0
5
No
Yes
Dry vessels/columns/lines
33)
End Loop
274
0.0
0
No
Yes
End vessel/column line wash
34)
Time Stamp
396
0.0
21
No
Yes
Record current time
35)
Go Sub
287
0.0
3
No
Yes
Deliver ammonia to start cleavage
36)
Clv Blk Flush
144
10.0
0
No
Yes
Flush ammonia from lower civ blocks
37)
Dep Xfer Rinse
151
5.0
0
No
Yes
Wash ammonia from lower civ blocks
C-12 Cycles, Procedures, and System Messages
Table C-4 Standard Cleavage Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
38)
Clv Blk Flush
FUNCTION
144
10.0
0
No
Yes
Dry lower cleavage blocks
Comments
39)
Wait
259
60.0
0
No
Yes
Wait while cleaving
40)
Wait
259
60.0
0
No
Yes
Wait while cleaving
41)
Wait
259
60.0
0
No
Yes
Wait while cleaving
42)
Set Pres Reg 10
310
0.0
4
No
Yes
Set regulator 10 to 4 psi
43)
Go Sub
287
0.0
1
No
Yes
Push fresh NH4 over cols for cleavage
44)
Wait
259
60.0
0
No
Yes
Wait while cleaving
45)
Wait
259
60.0
0
No
Yes
Wait while cleavinq
46)
Wait
259
60.0
0
No
Yes
Wait while cleavinq
47)
Wait
259
20.0
0
No
Yes
Wait while cleavinq
48)
Begin Loop
273
0.0
5
No
Yes
Begin cleavage loop
49)
Go Sub
287
0.0
1
No
Yes
Push fresh NH4 over cols for cleavage
50)
Wait
259
60.0
0
No
Yes
Wait while cleaving
51)
Wait
259
60.0
0
No
Yes
Wait while cleaving
52)
Wait
259
60.0
0
No
Yes
Wait while cleaving
53)
Wait
259
60.0
0
No
Yes
Wait while cleaving
54)
Wait
259
60.0
0
No
Yes
Wait while cleaving
55)
Wait
259
60.0
0
No
Yes
Wait while cleaving
56)
Wait
259
60.0
0
No
Yes
Wait while cleaving
57)
Wait
259
60.0
0
No
Yes
Wait while cleavina
58)
End Loop
274
0.0
0
No
Yes
End cleavage loop
59)
Time Stamp
396
0.0
22
No
Yes
Record current time
60)
Clv Wants Depro
284
0.0
0
No
Yes
Wait until deprotection coils are free
61)
Time Stamp
396
0.0
23
No
Yes
Record current time
62)
Set Coil Temp
260
0.0
0
No
Yes
Temp for transfer into coils (set in prefs)
63)
Go Sub
287
0.0
9
No
Yes
Drain/wash/dry of coils & blocks
64)
Time Stamp
396
0.0
24
No
Yes
Record current time
65)
Set Rmp Fxn Sns
265
0.0
10
No
Yes
Set ramp parms: # of total readings
66)
Set Rmp Fxn Trp
266
0.0
9
No
Yes
Set ramp parms: # of readings to trip
67)
Set Rmp Fxn Chk
267
0.0
7
No
Yes
Set ramp parms: # of readings to verify
68)
Set Rmp Fxn Dly
268
0.0
50
No
Yes
Set ramp parms: # of ms to delay verify
69)
Go Sub C
294
0.0
2
No
Yes
Prepare transfer to coil C
70)
Xfer Clv Col C
143
12.0
10
Yes
Yes
Ramping transfer from vessel C to coil C
71)
Go Sub B
293
0.0
2
No
Yes
Prepare transfer to coil B
72)
Xfer Clv Col B
142
12.0
10
Yes
Yes
Ramping transfer from vessel B to coil B
73)
Go Sub A
292
0.0
2
No
Yes
Prepare transfer to coil A
74)
Xfer Clv Col A
141
12.0
10
Yes
Yes
Ramping transfer from vessel A to coil A
75)
Start Depro Htr
170
0.0
0
No
Yes
Start depro heater (set in preferences)
76)
Time Stamp
396
0.0
25
No
Yes
Record current time
77)
Go Sub
287
0.0
5
No
Yes
Dry vessels/columns/lines
78)
Go Sub
287
0.0
3
No
Yes
Deliver ammonia for column strip
Cycles, Procedures, and System Messages C-13
Table C-4 Standard Cleavage Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
79)
Wait
FUNCTION
259
30.0
0
No
Yes
Wait while stripping column
80)
Wait
259
60.0
0
No
Yes
Wait while stripping column
81)
Set Pres Reg 10
310
0.0
4
No
Yes
Set regulator 10 to 4 psi
82)
Ammonia toWaste
115
1.0
0
No
Yes
Prime block with ammonia
83)
Ammonia to ClvA
112
1.0
0
No
Yes
Deliver ammonia to column A
84)
Ammonia to ClvB
113
1.0
0
No
Yes
Deliver ammonia to column B
85)
Ammonia to ClvC
114
1.0
0
No
Yes
Deliver ammonia to column C
86)
Clv Blk Flush
144
2.0
0
No
Yes
Flush ammonia from lower civ blocks
87)
Wait
259
30.0
0
No
Yes
Wait while stripping column
88)
Wait
259
60.0
0
No
Yes
Wait while stripping column
89)
Begin Loop
273
0.0
6
No
Yes
Begin column strip loop
90)
Go Sub
287
0.0
1
No
Yes
Gas push (for strip)
91)
Wait
259
30.0
0
No
Yes
Wait while stripping
92)
Wait
259
60.0
0
No
Yes
Wait while stripping
93)
End Loop
274
0.0
0
No
Yes
End column strip loop
94)
Go Sub
287
0.0
4
No
Yes
Drain & dry vessel/column/line
95)
Go Sub
287
0.0
5
No
Yes
Dry vessels/columns/lines
96)
Go Sub
287
0.0
7
No
Yes
Delivery of water to sensor
97)
Go Sub
287
0.0
6
No
Yes
Serial 5 psi gas bubble of vessel
98)
Go Sub
287
0.0
4
No
Yes
Drain vessel/column/line
99)
Go Sub
287
0.0
5
No
Yes
Dry vessels/columns/lines
100)
Go Sub
287
0.0
8
No
Yes
2:1 water/ACN vessel wash
101)
Go Sub
287
0.0
4
No
Yes
Drain & dry vessel/column/line
102)
Go Sub
287
0.0
5
No
Yes
Dry vessels/columns/lines
103)
Go Sub
287
0.0
8
No
Yes
2:1 water/ACN vessel wash
104)
Go Sub
287
0.0
4
No
Yes
Drain vessels/column/line
105)
Go Sub
287
0.0
5
No
Yes
Dry vessels/columns/lines
106)
Time Stamp
396
0.0
26
No
Yes
Record current time
107)
Goto End
290
0.0
0
No
Yes
Skip over subroutines to cycle end
108)
Sub Label
289
0.0
1
No
Yes
Gas push (for cleavage & strip)
109)
Clv Blk Flush
144
1.0
0
No
Yes
Flush lower cleavage blocks
110)
Press Line
138
2.0
0
No
Yes
Pressurize line to avoid-reverse flow
111)
Gas to ClvCol A
122
0.6
0
No
Yes
Push of fresh NH4 over col A for strip
112)
Clv Blk Flush
144
1.0
0
No
Yes
Flush lower cleavage blocks
113)
Press Line
138
2.0
0
No
Yes
Pressurize line to avoid reverse flow
114)
Gas to ClvCol B
123
0.6
0
No
Yes
Push of fresh NH4 over col B for strip
115)
Clv Blk Flush
144
1.0
0
No
Yes
Flush lower cleavage blocks
116)
Press Line
138
2.0
0
No
Yes
Pressurize line to avoid reverse flow
117)
Gas to ClvCol C
124
0.6
0
No
Yes
Push of fresh NH4 over col C for strip
118)
Return Sub
288
0.0
0
No
Yes
End of subroutine 1
119)
Sub Label
289
0.0
2
No
Yes
Prepare transfer to coils AIB/C
C-14 Cycles, Procedures, and System Messages
Comments
Table C-4 Standard Cleavage Cycle (v4.20) (continued)
NUM
TIME
MISC
SNS
SAFE
120)
STEP
Set Pres Reg 10
FUNCTION
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
Comments
121)
Dep Xfer Rinse
151
3.0
0
No
Yes
Water rinse for transfer line
122)
Dep Xfer Flush
152
20.0
0
No
Yes
Flush transfer line
123)
Set Pres Reg 1
301
0.0
12
No
Yes
Set regulator 1 to 12 psi
124)
DepUprBlk Rinse
154
3.0
0
No
Yes
Water rinse for upper depro block
125)
DepUprBlk Flush
221
5.0
0
No
Yes
Flush upper depro block
126)
DepLowBlk Flush
153
2.0
0
No
Yes
Flush lower depro block
127)
Set Pres Reg 10
310
0.0
5
No
Yes
Set regulator 10 to 5 psi
128)
Dep Xfer Flush
152
4.0
0
No
Yes
Reduce regulator 10 pressure
129)
Gas to Clv Cols
121
12.0
0
No
Yes
Consolidate sample into vessels
130)
Set Pres Reg 10
310
0.0
4
No
Yes
Starting transfer pressure
131)
Dep Xfer Flush
152
2.0
0
No
Yes
Reduce regulator 10 pressure
132)
Return Sub
288
0.0
0
No
Yes
End of subroutine 2
133)
Sub Label
289
0.0
3
No
Yes
Deliver ammonia for cleave/strip
134)
Ammonia toWaste
115
1.0
0
No
Yes
Prime block w/ ammonia
135)
Ammonia to Clvs
111
3.0
0
No
Yes
Start split delivery of NH4
136)
Ammonia to Clvs
111
15.0
0
Yes
Yes
Finish split delivery of NH4 to sensors
137)
Return Sub
288
0.0
0
No
Yes
End of subroutine 3
138)
Sub Label
289
0.0
4
No
Yes
Drain vessel/column/line
139)
Set Pres Reg 10
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
140)
Back Flush Clvs
131
6.0
0
No
Yes
Wet sensors for all active vessels
141)
Back Flush Clvs
131
45.0
0
Yes
Yes
Drain all vessels until sensors read dry
142)
Back Flsh Clv A
132
6.0
0
No
Yes
Complete drain of vessel A
143)
Back Flsh Clv B
133
6.0
0
No
Yes
Complete drain of vessel B
144)
Back Flsh Clv C
134
6.0
0
No
Yes
Complete drain of vessel C
145)
Begin Loop
273
0.0
2
No
Yes
Begin vessel drain/dry loop
146)
Gas to Clv Cols
121
1.0
0
No
Yes
Reverse drying
147)
Back Flush Clvs
131
4.0
0
No
Yes
Backflush drying
148)
End Loop
274
0.0
0
No
Yes
End vessel drain/dry loop
149)
Return Sub
288
0.0
0
No
Yes
End of subroutine 4
150)
Sub Label
289
0.0
5
No
Yes
Dry vessels/columns/lines
151)
Set Pres Reg 10
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
152)
Begin Loop
273
0.0
2
No
Yes
Begin drain/dry loop
153)
Back Flsh Clv A
132
5.5
0
No
Yes
Drain column A
154)
Back Flsh Clv B
133
5.5
0
No
Yes
Drain column B
155)
Back Flsh Clv C
134
5.5
0
No
Yes
Drain column C
156)
End Loop
274
0.0
0
No
Yes
End drain/dry loop
157)
Gas to Clv Cols
121
2.0
0
No
Yes
Reverse drying
158)
Back Flush Clvs
131
8.0
0
No
Yes
Backflush drying
159)
Return Sub
288
0.0
0
No
Yes
End of subroutine 5
160)
Sub Label
289
0.0
6
No
Yes
Serial 5 psi gas bubble of vessel
Cycles, Procedures, and System Messages C-15
Table C-4 Standard Cleavage Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
161)
Set Pres Reg 10
FUNCTION
310
0.0
5
No
Yes
Set regulator 10 to 5 psi
162)
Gas to ClvCol A
122
8.0
0
No
Yes
Bubble vessel A
163)
Gas to ClvCol B
123
8.0
0
No
Yes
Bubble vessel B
164)
Gas to ClvCol C
124
8.0
0
No
Yes
Bubble vessel C
165)
Return Sub
288
0.0
0
No
Yes
End of subroutine 6
166)
Sub Label
289
0.0
7
No
Yes
Delivery of water to sensor
167)
H2O to ClvWaste
130
1.0
0
No
Yes
Prime lower cleavage blocks w/ water
168)
H2O to Clv Cols
126
2.0
0
No
Yes
Start split delivery of water
169)
H2O to Clv Cols
126
15.0
0
Yes
Yes
Finish split delivery of water to sensor
170)
Return Sub
288
0.0
0
No
Yes
End of subroutine 7
171)
Sub Label
289
0.0
8
No
Yes
2:1 water/ACN vessel wash
172)
Set Pres Reg 10
310
0.0
6
No
Yes
Set regulator 10 to 6 psi
173)
ACN to Clv Cols
116
20.0
0
Yes
Yes
Deliver ACN to sensors
174)
Gas to Clv Cols
121
15.0
0
Yes
Yes
Push ACN until sensors read dry
175)
Gas to Clv Cols
121
3.0
0
No
Yes
Consolidate ACN into vessels
176)
Set Pres Reg 10
310
0.0
8
No
Yes
Set regulator 10 to 8 psi
177)
Begin Loop
273
0.0
2
No
Yes
Begin water delivery loop
178)
H2O to Clv Cols
126
15.0
0
Yes
Yes
Deliver water to sensors
179)
Gas to Clv Cols
121
15.0
0
Yes
Yes
Push water until sensors read dry
180)
Gas to Clv Cols
121
3.0
0
No
Yes
Consolidate water into vessels
181)
End Loop
274
0.0
0
No
Yes
End water delivery loop
182)
Set Pres Reg 10
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
183)
Gas to Clv Cols
121
10.0
0
No
Yes
Bubble/wash of water/ACN in vessels
184)
Return Sub
288
0.0
0
No
Yes
End of subroutine 8
185)
Sub Label
289
0.0
9
No
Yes
Drain/wash/dry of coils & blocks
186)
Engage All Cols
300
0.0
0
No
Yes
Force all columns to be active
187)
Set Pres Reg 1
301
0.0
12
No
Yes
Set regulator 1 to 12 psi
188)
Gas to Coil A
156
15.0
0
No
Yes
Drain coil A
189)
Gas to Coil B
157
15.0
0
No
Yes
Drain coil B
190)
Gas to Coil C
158
15.0
0
No
Yes
Drain coil C
191)
H20 to Coil A
163
8.0
0
No
Yes
Wash coil A
192)
H20 to Coil B
164
8.0
0
No
Yes
Wash coil B
193)
H20 to Coil C
165
8.0
0
No
Yes
Wash coil C
194)
Gas to Coil A
156
15.0
0
No
Yes
Dry coil A
195)
Gas to Coil B
157
15.0
0
No
Yes
Dry coil B
196)
Gas to Coil C
158
15.0
0
No
Yes
Dry coil C
197)
DepUprBlk Rinse
154
3.0
0
No
Yes
Water rinse for upper depro block
198)
DepUprBlk Flush
221
5.0
0
No
Yes
Flush upper depro block
199)
Set Pres Reg 10
310
0.0
12
No
Yes
Set regulator 10 to 12 psi
200)
H2O to ClvWaste
130
3.0
0
No
Yes
Wash lower cleavage blocks w/ water
201)
Clv Blk Flush
144
5.0
0
No
Yes
Flush lower cleavage blocks
C-16 Cycles, Procedures, and System Messages
Comments
Table C-4 Standard Cleavage Cycle (v4.20) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
202)
Select Act Cols
FUNCTION
283
0.0
0
No
Yes
Select only cols available for chemistry
Comments
203)
Return Sub
288
0.0
0
No
Yes
End of subroutine 8
204)
End of Cycle
272
0.0
0
No
Yes
Marks end of cycle and all subroutines
Cycles, Procedures, and System Messages C-17
The Standard Purification Cycle
Purification Cycle The Purification cycle processes/reactions needed for oligo production are illustrated
Summary in the following flow chart (Figure C-3) and the step numbers corresponding to these
activities are listed in Table C-5. Table C-6 contains a listing of the standard
purification cycle:
Figure C-3 Purification Cycle Flow Chart
C-18 Cycles, Procedures, and System Messages
Table C-5 Summary of Purification Cycle
Step Range
1 - 16
17 - 18
Description
Test for Active Columns
♦
Purification owns the deprotection coils.
♦
Test for oligos that have ailed leak test in synthesis or cleavage
cycles.
♦
If synthesis or cleavage modules failed leak test, then (a) wait for
Syn and Clv modules to complete leak testing, (b) advance the
sample collector 3 times for the next set of oligos, (c) drop the coil
temperature to the transfer IN setting f(x) 260 and (d) give the
depro coils to the cleavage module. Go to Cycle end.
♦
If synthesis or cleavage modules passed leak test, then close the
purification jaw.
Start Here if Columns Active, Leak Test
♦
19 - 50
51 - 58
Start Purification Cycle or Recover Sample in Coils
♦
If leak test passes, then go to start of purification cycle at step 51.
♦
If leak test fails, restore active columns that were originally set
from synthesis and execute the recovery of crude oligos from the
coils.
♦
Set ramping function parameters. Wait for deprotection of crude
oligos to complete. Drop the coil temperature to the transfer out
setting. Set the coils to the transfer in temperature for the next
row of oligos.
♦
Crude A to UV. Clean the purification blocks, transfer line, UV cell
and sample collector delivery line. Move the sample collector
(SC) rack to the load (close) position for the next sample. Lower
the SC needle. Execute the “crude to UV” ramping function and
transfer the crude oligo into the SC tube.
♦
Crude C to UV. Clean the purification blocks, transfer line, UV cell
and sample collector delivery line. Move the sample collector
(SC) rack to the load (close) position for the next sample. Lower
the SC needle Execute the “crude to UV” ramping function and
transfer the crude oligo into the SC tube.
♦
Wash and dry purification blocks, transfer line, UV cell and SC
delivery line. Advance the SC for the next sample and open SC
(move to front).
♦
Give cleavage the depro coils and end recovery routine.
Start Here if Purification Passed Leak Test, Wash and Dry Purification
Module
♦
59 - 60
Close the purification jaw, begin leak test and check for pass or
fail.
Set regulator 3 to 12 psi. Flush and wash lower pur blocks. Drain
and dry pur module. Wash vessels with 2:1 water/ACN. Drain and
dry pur module
Test for Crude or Purified Oligos
♦
Activate only the columns to be purified.
♦
Check for the columns to be purified. If yes, then go to step 61. If
no, then skip to step 105 “Select Act Cols”.
Cycles, Procedures, and System Messages C-19
Table C-5 Summary of Purification Cycle (continued)
Step Range
61 - 68
Description
Pre TFA Scrub, ACN/Water Wash of Pur Module
♦
69 - 70
TFA Scrub of Columns to be Used for Purification
♦
71 - 85
86 - 104
105
106 -122
123 - 124
125 - 126
127 - 138
C-20 Cycles, Procedures, and System Messages
Two ACN (Go Sub 5) and water (Go Sub 4) washes of the
purification module. Drain and dry (Go Sub3) of the purification
module.
The OneStep™ columns that are selected to be purified are
scrubbed with 3% TFA (Go Sub 9) to remove any product that
was not cleaved during the cleavage portion of the cleavage
cycle.
Post TFA Scrub, ACN/Water Wash of Pur Module
♦
Two AN washes of Pur module (Go Sub 5). Split parallel delivery
of ACN to Pur Cols and split serial delivery of Water to Pur Cols.
♦
Loop the split deliveries two times. Water wash of pur module
(Go Sub 4). Drain and Dry pur module (Go Sub 3).
Column Prep for Oligos to be Purified
♦
Split parallel delivery of TEAA to pur sensors. Flush upper pur
block. Serial push of TEAA over columns into the vessels. Serial
backflush of TEAA from the vessels over the columns out to
waste.
♦
Three loops of a serial backflush to drain and dry the columns.
Clean lower; purification block (Go Sub 10).
Select All Columns for Transfer of Oligos from Coils into the
Purification Vessels
Transfer Oligos from the Deprotection Coils into the Purification
Vessels
♦
Set ramping function parameters (Go Sub 11).
♦
Purification module waits for deprotection to complete.
♦
Drop the coil temp. to the transfer out temperature.
♦
Purification block and transfer line are washed before the coil to
pur column transfer.
♦
The coil contents are transferred into the pur vessels in column
order C, B, A. The pur block and transfer line are rinsed and dried
after the ramping transfer.
Cleavage Owns Depro Coils
♦
The lower purification is rinsed and dried with water.
♦
Cleavage owns the deprotection coils.
Test for Crude or Purified Oligos
♦
Activate only the columns to be purified.
♦
Check for the columns to be purified. If yes, then go to step 127.
♦
If no, then skip to step 162 “Select Act Cols”.
Sample Neutralization Loop
♦
Consolidate the crude sample in the vessels and dry the
purification sensors. Flush lower pur block.
♦
Two parallel sensored deliveries of Acetic Acid to the columns.
Table C-5 Summary of Purification Cycle (continued)
Step Range
139 - 142
143 - 148
149 - 152
Description
Bonding of Trityl ON Oligos to Columns
♦
Gently consolidate sample into the purification vessels.
♦
Start serial backflush over columns at 6 psi, column order A, B, C
(Go Sub 7). The serial backflush will ramp the pressure if the
purification sensor does not read “dry” at the initial pressure
setting of 6 psi.
Cleavage/Detritylation of Bound Oligo from Column
♦
Wash and dry column with water.
♦
Start detritylation by delivering 3% TFA to the upper pur sensors.
Two loops of (Go Sub 9, TFA push/waits over the column.
Post Cleavage/Detritylation, Wash of Purification Module
♦
153 - 161
162
Three loops of water washing of purification module (Go Sub 4),
followed by a drain and dry for purification module.
Elution of Oligos from Column into Vessels with 20% ACN
♦
Parallel split delivery of 0% ACN to Pur Cols that elutes the oligos
form the column support into the vessels. Set regulator 3 to 4 psi.
♦
Gently consolidate and bubble purified oligos in the purification
vessels.
Select All Columns for Transfer of Oligos from the Purification Vessels
to UV Cell
Cycles, Procedures, and System Messages C-21
Table C-5 Summary of Purification Cycle (continued)
Step Range
163 - 191
Description
UV Quantitation and Sample Collection
Position A:
♦
Take UV baseline reading (Go Sub 1).
♦
Wash the UV cell and SC delivery line (Go Sub 12).
♦
Advance SC to the back (closed position) for next sample, lower
SC needle into tube.
♦
Consolidate the sample into the vessel. Ramping sensored
transfer of oligo, crude or purified, into the UV cell.
♦
Deliver ACN/water into the pur vessel (Go Sub 14).
♦
Quantitate oligo and deliver into SC vial (Go Sub 15).
Position B:
♦
Take UV baseline reading (Go Sub 1).
♦
Wash the UV cell and SC delivery line (Go Sub 12).
♦
Advance SC to the next position and lower the SC needle into the
collection vial.
♦
Consolidate the sample into the vessel. Ramping sensored
transfer of oligo, crude or purified, into the UV ell.
♦
Deliver ACN/water into the pur vessel (Go Sub 14).
♦
Quantitate oligo and deliver into SC vial (Go Sub 16).
Position C:
192 - 199
C-22 Cycles, Procedures, and System Messages
♦
Take UV baseline reading (Go Sub 1).
♦
Wash the UV cell and SC delivery line (Go Sub 12).
♦
Advance SC to the next position and lower the SC needle into the
collection vial.
♦
Consolidate the sample into the vessel. Ramping sensored
transfer of oligo, crude or purified, into the UV ell.
♦
Deliver ACN/water into the pur vessel (Go Sub 14).
♦
Quantitate oligo and deliver into SC vial (Go Sub 17). Wash UC
cell and SC delivery line.
♦
Advance SC to the next position and move the SC to the front
(open position).
Post Cycle, Purification Module Wash
♦
Push ACN/water into the purification vessels until the sensors are
dry.
♦
Wash vessel by bubbling contents. Drain and dry pur module.
♦
2:1 water/AN wash of pur vessels (Go Sub 8). Drain and dry
purification module (Go Sub 3).
Purification Cycle The annotated listing below is provided to aid you in understanding ABI 3948 system
Listing cleavage and deprotection:
Table C-6 Standard Purification Cycle (v4.52)
STEP
FUNCTION
NUM
TIME
MIS
SNS
SAFE
Comments
1)
Begin of Cycle
271
0
0
No
Yes
Marks beginning of cycle
2)
Pur Owns Depro
285
0.0
0
No
Yes
Purification controls deprotection coils
3)
If Any Act Cols
291
0.0
0
No
Yes
Test for oligos that failed in Syn or Clv
4)
Else
280
0.0
0
No
Yes
If ok, do nothing, else advance SC
5)
Wait
259
60.0
0
No
Yes
Wait for syn/civ module leak testing
6)
Wait
259
60.0
0
No
Yes
Wait for syn/civ module leak testing
7)
Wait
259
60.0
0
No
Yes
Wait for syn/civ module leak testing
8)
SC Next
244
0.0
0
No
Yes
Advance sample collector for bad row
9)
SC Next
244
0.0
0
No
Yes
Advance samPIe collector for bad row
10)
SC Next
244
0.0
0
No
Yes
Advance sample collector for bad row
11)
SC Open
245
0.0
0
No
Yes
Bring sample collector rack to the front
12)
Wait Coil Temp
261
0.0
0
No
Yes
Drop coil temp to transfer OUT temp
13)
Set Coil Temp
260
0.0
0
No
Yes
Set coil temp to transfer IN temp
14)
Clv Owns Depro
286
0.0
0
No
Yes
Gives deprotection coils to cleavage
15)
Goto End
290
0.0
0
No
Yes
Skip over subroutines to cycle end
16)
Endif
281
0.0
0
No
Yes
End of test for bad row
17)
Pur Jaw Close
236
0.0
0
No
Yes
CIose pur jaw, start pur module leak test
18)
If Any Act Cols
291
0.0
0
No
Yes
If test passes, do nothing
19)
Else
280
0.0
0
No
Yes
If test fails, recover crudes from coils
20)
Revive Act Cols
319
0.0
0
No
Yes
Select column that produced crudes
21)
Go Sub
287
0.0
11
No
Yes
Set ramping function parameters
22)
Depro Htr Wait
169
0.0
0
No
Yes
Wait for deDrotection to comDIete
23)
Wait Coil Temp
261
0.0
0
No
Yes
Drop coil temp to transfer OUT temp
24)
Set Coil Temp
260
0.0
0
No
Yes
Set coil temp to transfer IN temp
25)
Go Sub
287
0.0
2
No
Yes
CIean blocks and pur transfer line
26)
Go Sub
287
0.0
1
No
Yes
Take UV baseline reading
27)
Go Sub
287
0.0
12
No
Yes
Wash UV cell and SC delivery line
28)
SC Close
246
0.0
0
No
Yes
SC rack to load position for next sample
29)
SC Needle Down
248
0.0
0
No
Yes
Lower needle into collection vial
30)
Go Sub A
292
0.0
6
No
Yes
Recovery: crude coils to UV
31)
Xfer UV to SC
215
15.0
0
No
Yes
Transfer oligo to SC
32)
Go Sub
287
0.0
2
No
Yes
CIean blocks and pur transfer line
33)
Go Sub
287
0.0
1
No
Yes
Take UV baseline reading
34)
Go Sub
287
0.0
12
No
Yes
Wash UV cell and SC delivery line
35)
SC Next
244
0.0
0
No
Yes
Advance SC for next sample
36)
Go Sub B
293
0.0
6
No
Yes
Recovery: crude coils to UV
37)
Xfer UV to SC
215
15.0
0
No
Yes
Transfer oligo to SC
38)
Go Sub
287
0.0
2
No
Yes
CIean blocks and pur transfer line
Cycles, Procedures, and System Messages C-23
Table C-6 Standard Purification Cycle (v4.52) (continued)
STEP
NUM
TIME
MIS
SNS
SAFE
39)
Go Sub
FUNCTION
287
0.0
1
No
Yes
Take UV baseline reading
40)
Go Sub
287
0.0
12
No
Yes
Wash UV cell and SC delivery line
41)
SC Next
244
0.0
0
No
Yes
Advance SC for next sample
42)
Go Sub C
294
0.0
6
No
Yes
Recovery: crude coils to UV
43)
Xfer UV to SC
215
15.0
0
No
Yes
Transfer oligo to SC
44)
Go Sub
287
0.0
2
No
Yes
CIean blocks and our transfer line
45)
Go Sub
287
0.0
12
No
Yes
Wash UV cell and SC delivery line
46)
SC Next
244
0.0
0
No
Yes
Advance SC for next sample
47)
SC Open
245
0.0
0
No
Yes
SC rack to front
48)
Clv Owns Depro
286
0.0
0
No
Yes
Cleavage owns the deprotection coils
49)
Goto End
290
0.0
0
No
Yes
End of recovery of crudes routine
50)
Endif
281
0.0
0
No
Yes
To reach here, leak test passed
51)
Time Stamp
396
0.0
30
No
Yes
Time stamp
52)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
53)
PurLowBlk Flush
218
5.0
0
No
Yes
Flush lower pur block, may be wet
54)
ACN to PurWaste
177
5.0
0
No
Yes
ACN rinse of lower pur block
55)
PurLowBlk Flush
218
5.0
0
No
Yes
Flush lower pur block
56)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
57)
Go Sub
287
0.0
8
No
Yes
2:1 water/ACN vessel wash
58)
Go Sub
287
0.0
3
No
Yes
Drain & dry,vessels/columns/lines
59)
Select Pur Cols
282
0.0
0
No
Yes
Activate only the columns to be purified
60)
If Any Act Cols
291
0.0
0
No
Yes
Check here for any columns to be purified
61)
Go Sub
287
0.0
5
No
Yes
ACN wash & bubble
62)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
63)
Go Sub
287
0.0
5
No
Yes
ACN wash & bubble
64)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
65)
Go Sub
287
0.0
4
No
Yes
Water wash & bubble
66)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
67)
Go Sub
287
0.0
4
No
Yes
Water wash & bubble
68)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
69)
Go Sub
287
0.0
9
No
Yes
Deliver TFA Scrub and drain
70)
Go Sub
287
0.0
9
No
Yes
Deliver TFA Scrub and drain
71)
Go Sub
287
0.0
5
No
Yes
ACN wash & bubble
72)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
73)
Go Sub
287
0.0
5
No
Yes
ACN wash & bubble
74)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
75)
Begin Loop
273
0.0
2
No
Yes
Begin ACN/water wash loop
76)
ACN to Pur Cols
173
2.2
0
No
Yes
Start split delivery of ACN
77)
ACN to Pur Cols
173
15.0
0
Yes
Yes
Finish split delivery of ACN to sensor
78)
H2O to PurCol A
184
5.0
0
No
Yes
Water delivery to vessel A
79)
H2O to PurCol B
185
5.0
0
No
Yes
Water delivery to vessel B
C-24 Cycles, Procedures, and System Messages
Comments
Table C-6 Standard Purification Cycle (v4.52) (continued)
STEP
NUM
TIME
MIS
SNS
SAFE
80)
H2O to PurCol C
FUNCTION
186
5.0
0
No
Yes
Water delivery to vessel C
Comments
81)
Gas to Pur Cols
203
15.0
0
No
Yes
Consolidate ACN/water into vessels
82)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
83)
End Loop
274
0.0
0
No
Yes
End ACN/water wash loop
84)
Go Sub
287
0.0
4
No
Yes
Water wash & bubble
85)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
86)
Set Pres Reg 3
303
0.0
9
No
Yes
Set regulator 3 to 9 psi
87)
TEAA to Waste
182
5.0
0
No
Yes
Prime block w/ TEAA
88)
TEAA to PurCols
178
25.0
0
No
Yes
Start split delivery of TEAA
89)
TEAA to PurCols
178
45.0
0
Yes
Yes
Finish split delivery of TEAA to sensors
90)
PurUprBlk Flush
219
2.0
0
No
Yes
Flush upper pur block
91)
Gas to PurCol A
204
10.0
0
No
Yes
Push TEAA over column into vessel A
92)
Gas to PurCol B
205
10.0
0
No
Yes
Push TEAA over column into vessel B
93)
Gas to PurCol C
206
10.0
0
No
Yes
Push TEAA over column into vessel C
94)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
95)
Back Flsh Pur A
208
20.0
0
No
Yes
Drain TEAA from vessel A
96)
Back Flsh Pur B
209
20.0
0
No
Yes
Drain TEAA from vessel B
97)
Back Flsh Pur C
210
20.0
0
No
Yes
Drain TEAA from vessel C
98)
Begin Loop
273
0.0
3
No
Yes
Begin TEAA drain/dry loop
99)
Back Flsh Pur A
208
10.0
0
No
Yes
Drain TEAA from vessel A
100)
Back Flsh Pur B
209
10.0
0
No
Yes
Drain TEAA from vessel B
101)
Back Flsh Pur C
210
10.0
0
No
Yes
Drain TEAA from vessel C
102)
End Loop
274
0.0
0
No
Yes
End TEAA drain/dry loop
103)
Go Sub
287
0.0
10
No
Yes
Clean lower purification block
104)
Endif
281
0.0
0
No
Yes
End of purification column prep
105)
Select Act Cols
283
0.0
0
No
Yes
Select crude and purified columns
106)
Go Sub C
294
0.0
2
No
Yes
Clean blocks and pur transfer line
107)
Go Sub
287
0.0
11
No
Yes
Set ramping function parameters
108)
Time Stamp
396
0.0
31
No
Yes
Time stamp
109)
Depro Htr Wait
169
0.0
0
No
Yes
Wait for deprotection to complete
110)
Time Stamp
396
0.0
32
No
Yes
Time stamp
111)
Wait Coil Temp
261
0.0
0
No
Yes
Drop coil temp to transfer OUT temp
112)
Set Coil Temp
260
0.0
0
No
Yes
Set coil temp to transfer IN temp
113)
Time Stamp
396
0.0
33
No
Yes
Time stamp
114)
Xfer Coil C
168
12.0
1
Yes
No
Ramping transfer from coil C to pur sensor
115)
Xfer Coil C
168
20.0
0
No
No
Push sample from sensor to vessel
116)
Go Sub B
293
0.0
2
No
Yes
Clean blocks and pur transfer line
117)
Xfer Coil B
167
12.0
1
Yes
No
Ramping transfer from coil B to pur sensor
118)
Xfer Coil B
167
20.0
0
No
No
Push sample from sensor to vessel
119)
Go Sub A
292
0.0
2
No
Yes
Clean blocks and pur transfer line
120)
Xfer Coil A
166
12.0
1
Yes
No
Ramping transfer from coil A to pur sensor
Cycles, Procedures, and System Messages C-25
Table C-6 Standard Purification Cycle (v4.52) (continued)
STEP
NUM
TIME
MIS
SNS
SAFE
121)
Xfer Coil A
FUNCTION
166
20.0
0
No
No
Push sample from sensor to vessel
122)
Time Stamp
396
0.0
34
No
Yes
Time stamp
123)
Clv Owns Depro
286
0.0
0
No
Yes
Cleavage owns the depro coils
124)
Go Sub
287
0.0
10
No
Yes
Clean lower purification block
125)
Select Pur Cols
282
0.0
0
No
Yes
Select only the purified oligos
126)
If Any Act Cols
291
0.0
0
No
Yes
Check if there are any oligos to purify
127)
Set Pres Reg 3
303
0.0
9
No
Yes
Set regulator 3 to 9 psi
128)
Gas to Pur Cols
203
60.0
0
No
Yes
Extra drying of purification sensors
129)
Set Pres Reg 3
303
0.0
5
No
Yes
Set regulator 3 to 5 psi
130)
PurLowBlk Flush
218
2.0
0
No
Yes
Fiush lower pur block
131)
Begin Loop
273
0.0
2
No
Yes
Begin neutralization loop
132)
AcAcid to Waste
202
2.0
0
No
Yes
Prime block with acetic acid
133)
AcAcid to Purs
198
3.0
0
No
Yes
Start split delivery of acetic acid
134)
AcAcid to Purs
198
30.0
0
Yes
Yes
Finish split delivery of acetic acid to sensor
135)
Gas to Pur Cols
203
25.0
0
No
Yes
Mix samples in vessels and dry sensors
136)
ACN to PurWaste
177
2.0
0
No
Yes
ACN rinse of lower pur block
137)
PurLowBlk Flush
218
5.0
0
No
Yes
Flush lower pur block
138)
End Loop
274
0.0
0
No
Yes
End neutralization loop
139)
Gas to Pur Cols
203
15.0
0
No
Yes
Consolidate samples in vessels
140)
Go Sub A
292
0.0
7
No
Yes
Bind oligo to column A
141)
Go Sub B
293
0.0
7
No
Yes
Bind oligo to column B
142)
Go Sub C
294
0.0
7
No
Yes
Bind oligo to column C
143)
Go Sub
287
0.0
4
No
No
No Water wash & bubble
144)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
145)
Go Sub
287
0.0
4
No
Yes
Water wash & bubble
146)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
147)
Go Sub
287
0.0
9
No
Yes
Deliver TFA Detritylation
148)
Go Sub
287
0.0
9
No
Yes
Deliver TFA Detritylation
149)
Begin Loop
273
0.0
3
No
No
Begin water line/column/vessel wash loop
150)
Go Sub
287
0.0
4
No
No
Water wash & bubble
151)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
152)
End Loop
274
0.0
0
No
No
End water Erie/column/vessels wash loop
153)
20%ACN to Waste
197
4.0
0
No
Yes
Prime lower pur block w/ 20% ACN
154)
20%ACN to Purs
193
6.0
0
No
Yes
Start split delivery of 20% ACN
155)
20%ACN to Purs
193
15.0
0
Yes
Yes
Finish split deiiverv of 20% ACN, sensor
156)
Go Sub
287
0.0
10
No
Yes
Clean lower purification block
157)
Set Pres Reg 3
303
0.0
4
No
Yes
Set regulator 3 to 4 psi for elusion
158)
PurLowBlk Flush
218
2.0
0
No
Yes
Flush lower pur block
159)
Gas to Pur Cols
203
10.0
0
No
Yes
Elution: push 20% ACN to vessels
160)
Endif
281
0.0
0
No
Yes
End of purification
161)
Time Stamp
396
0.0
35
No
Yes
Time stamp
C-26 Cycles, Procedures, and System Messages
Comments
Table C-6 Standard Purification Cycle (v4.52) (continued)
NUM
TIME
MIS
SNS
SAFE
162)
STEP
Select Act Cols
FUNCTION
283
0.0
0
No
Yes
Select all crudes and purifieds
Comments
163)
Go Sub A
292
0.0
1
No
Yes
Take UV baseline reading
164)
Go Sub A
292
0.0
12
No
Yes
Wash UV cell and SC delivery line
165)
SC Close
246
0.0
0
No
Yes
SC rack to back for next sample
166)
SC Needle Down
248
0.0
0
No
Yes
Lower needle into collection vial
167)
Gas to PurCol A
204
7.0
0
No
Yes
Consolidate sample into vessel A
168)
Pur A to UV
211
10.0
3
Yes
Yes
Ramping delivery of A to UV sensor
169)
Pur A to UV
211
20.0
0
No
Yes
Finish delivery of A to UV cell
170)
Go Sub A
292
0.0
14
No
Yes
Deliver ACN/water to vessels
171)
Go Sub A
292
0.0
15
No
Yes
Quantitate & collect for sample A
172)
Go Sub B
293
0.0
1
No
Yes
Take UV baseline reading
173)
Go Sub B
293
0.0
12
No
Yes
Wash UV cell and SC delivery line
174)
SC Next
244
0.0
0
No
Yes
Advance SC for next sample
175)
Gas to PurCol B
205
7.0
0
No
Yes
Consolidate sample into vessel B
176)
Pur B to UV
212
10.0
3
Yes
Yes
Ramping delivery of B to UV sensor
177)
Pur B to UV
212
20.0
0
No
Yes
Finish delivery of B to UV cell
178)
Go Sub B
293
0.0
14
No
Yes
Deliver ACN/water to vessels
179)
Go Sub B
293
0.0
16
No
Yes
Quantitate & collect for sample B
180)
Go Sub C
294
0.0
1
No
Yes
UV baseline reading
181)
Go Sub C
294
0.0
12
No
Yes
Wash UV cell and SC delivery line
182)
SC Next
244
0.0
0
No
Yes
Advance SC for next sample
183)
Gas to PurCol C
206
7.0
0
No
Yes
Consolidate sample into vessel C
184)
Pur C to UV
213
10.0
3
Yes
Yes
Ramping delivery of C to UV sensor
185)
Pur C to UV
213
20.0
0
No
Yes
Finish delivery of C to UV cell
186)
Go Sub C
294
0.0
14
No
Yes
Deliver ACN/water to vessels
187)
Go Sub C
294
0.0
17
No
Yes
Quantitate & collect for sample C
188)
Go Sub
287
0.0
12
No
Yes
Wash UV cell and SC delivery line
189)
SC Next
244
0.0
0
No
Yes
Advance SC for next sample
190)
SC Open
245
0.0
0
No
Yes
SC rack to front
191)
Time Stamp
396
0.0
36
No
Yes
Time stamp
192)
Set Pres Reg 3
303
0.0
5
No
Yes
Set regulator 3 to 5 psi
193)
Gas to Pur Cols
203
45.0
0
Yes
Yes
Push contents into vessels until sensor dry
194)
Gas to Pur Cols
203
10.0
0
No
Yes
Bubble contents in vessels
195)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
196)
Go Sub
287
0.0
8
No
Yes
2:1 water/ACN vessel wash
197)
Go Sub
287
0.0
3
No
Yes
Drain & dry vessels/columns/lines
198)
Time Stamp
396
0.0
37
No
Yes
Time stamp
199)
Goto End
290
0.0
0
No
Yes
Skip over subroutines to cycle end
200)
Sub Label
289
0.0
1
No
Yes
Take UV baseline reading
201)
Set Pres Reg 3
303
0.0
6
No
Yes
Set regulator 3 to 6 psi
202)
SC Waste
242
0.0
0
No
Yes
Send the SC to the waste
Cycles, Procedures, and System Messages C-27
Table C-6 Standard Purification Cycle (v4.52) (continued)
STEP
NUM
TIME
MIS
SNS
SAFE
203)
H2O to PurWaste
FUNCTION
187
5.0
0
No
Yes
Water rinse for lower purification block
204)
H2O to UV
214
5.0
0
No
Yes
Water rinse of UV cell
205)
SC Block Flush
222
5.0
0
No
Yes
Flush lower pur blocks and UV cell dry
206)
Wait
259
10.0
0
No
Yes
Wait/soak
207)
UV Reading
216
2.0
0
No
Yes
Take baseline UV reading (misc=0)
208)
Flsh UV toWaste
217
6.0
0
No
Yes
Flush UV cell dry
209)
H2O to UV
214
1.0
0
No
Yes
Water rinse of UV cell
210)
SC Block Flush
222
10.0
0
No
Yes
Flush lower pur blocks and UV cell dry
211)
Xfer UV to SC
215
10.0
0
No
Yes
Flush UV cell to SC waste
212)
Return Sub
288
0.0
0
No
Yes
End of subroutine 1
213)
Sub Label
289
0.0
2
No
Yes
Clean blocks and pur transfer line
214)
Set Pres Reg 1
301
0.0
12
No
Yes
Set regulator 1 to 12 psi
215)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
216)
ACN to PurWaste
177
0.5
0
No
Yes
ACN rinse of lower pur block
217)
PurLowBlk Flush
218
3.0
0
No
Yes
Flush lower pur block
218)
Pur Xfer Rinse
171
2.0
0
No
Yes
Water rinse pur transfer line
219)
Pur Xfer Flush
172
15.0
0
No
Yes
Flush of pur transfer line
220)
Set Pres Reg 1
301
0.0
8
No
Yes
Pur to UV starting pressure
221)
DepLowBlk Flush
153
2.0
0
No
No
Flush lower depro block
222)
Return Sub
288
0.0
0
No
No
End of subroutine 2.
223)
Sub Label
289
0.0
3
No
Yes
Drain & dry vessels/columns/lines
224)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
225)
Back Flush Purs
207
6.0
0
No
Yes
Wet sensors for all active vessels
226)
Back Flush Purs
207
45.0
0
Yes
Yes
Drain all vessels until sensors read dry
227)
Back Flsh Pur A
208
6.0
0
No
Yes
Complete drain of vessel A
228)
Back Flsh Pur B
209
6.0
0
No
Yes
Complete drain of vessel B
229)
Back Flsh Pur C
210
6.0
0
No
Yes
Complete drain of vessel C
230)
Begin Loop
273
0.0
2
No
Yes
Begin vessel drain/dry loop
231)
Gas to Pur Cols
203
1.0
0
No
Yes
Reverse drying -
232)
Back Flush Purs
207
4.0
0
No
Yes
Backflush drying
233)
End Loop
274
0.0
0
No
Yes
End vessel drain/dry loop
234)
Return Sub
288
0.0
0
No
Yes
End of subroutine 3
235)
Sub Label
289
0.0
4
No
Yes
Water wash & bubble
236)
Set Pres Reg 3
303
0.0
9
No
Yes
Set regulator 3 to 9 psi
237)
H2O to Pur Cols
183
3.0
0
No
Yes
Start split delivery of water
238)
H2O to Pur Cols
183
15.0
0
Yes
Yes
Finish split delivery of water to sensor
239)
Gas to Pur Cols
203
15.0
0
Yes
Yes
Push water to vessel until sensor dry
240)
Gas to Pur Cols
203
6.0
0
No
Yes
Bubble/wash purification columns
241)
Return Sub
288
0.0
0
No
Yes
End of subroutine 4
242)
Sub Label
289
0.0
5
No
Yes
ACN wash & bubble
243)
Set Pres Reg 3
303
0.0
4
No
Yes
Set regulator 3 to 4 psi
C-28 Cycles, Procedures, and System Messages
Comments
Table C-6 Standard Purification Cycle (v4.52) (continued)
NUM
TIME
MIS
SNS
SAFE
244)
STEP
ACN to Pur Cols
FUNCTION
173
4.0
0
No
Yes
Start split delivery of ACN
Comments
245)
ACN to Pur Cols
173
15.0
0
Yes
Yes
Finish split delivery of ACN to sensor
246)
Gas to Pur Cols
203
15.0
0
Yes
Yes
Push ACN to vessel until sensor dry
247)
Gas to Pur Cols
203
6.0
0
No
Yes
Bubble/wash purification columns
248)
Return Sub
288
0.0
0
No
Yes
End of subroutine 5
249)
Sub Label
289
0.0
6
No
Yes
Recovery: crude coils to UV
250)
Crude A to UV
135
12.0
1
Yes
Yes
Transfer coils contents to UV sensor
251)
Crude A to UV
135
20.0
0
No
Yes
Push coils contents into cuvette
252)
Crude B to UV
136
12.0
1
Yes
Yes
Transfer coil B contents to UV sensor
253)
Crude B to UV
136
20.0
0
No
Yes
Push coil B contents into cuvette
254)
Crude C to UV
137
12.0
1
Yes
Yes
Transfer coil C contents to UV sensor
255)
Crude C to UV
137
20.0
0
No
Yes
Push coil C contents into cuvette
256)
Return Sub
288
0.0
0
No
Yes
End of subroutine 6
257)
Sub Label
289
0.0
7
No
Yes
Bind oligo to column A/B/C
258)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi .
259)
ACN to PurWaste
177
2.0
0
No
Yes
ACN rinse of lower pur block
260)
PurLowBlk Flush
218
5.0
0
No
Yes
Flush lower pur block
261)
Set Pres Reg 3
303
0.0
6
No
Yes
Set regulator 3 to 6 psi
262)
Back Flush Purs
207
15.0
0
No
Yes
Wet sensors for all active vessels
263)
Back Flush Purs
207
45.0
3
Yes
Yes
Bind vessel contents until sensor read dry
264)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
265)
Back Flush Purs
207
15.0
0
No
Yes
Complete vessel drain
266)
Return Sub
288
0.0
0
No
Yes
End of subroutine 7
267)
Sub Label
289
0.0
8
No
Yes
2:1 water/ACN vessel wash
268)
Set Pres Reg 3
303
0.0
6
No
Yes
Set regulator 3 to 6 psi
269)
ACN to Pur Cols
173
4.0
0
No
Yes
Start split delivery of ACN to columns
270)
ACN to Pur Cols
173
15.0
0
Yes
Yes
Split delivery of ACN to sensors
271)
Gas to Pur Cols
203
15.0
0
Yes
Yes
Push ACN to vessels until sensor reads dry
272)
Gas to Pur Cols
203
3.0
0
No
Yes
Bubble contents in vessel
273)
Set Pres Reg 3
303
0.0
8
No
Yes
Set regulator 3 to 8 psi
274)
Begin Loop
273
0.0
2
No
Yes
Begin water wash loop
275)
H2O to Pur Cols
183
4.0
0
No
Yes
Start split delivery of water to column
276)
H2O to Pur Cols
183
15.0
0
Yes
Yes
Finish split delivery of water to sensor
277)
Gas to Pur Cols
203
15.0
0
Yes
Yes
Push water to vessel until sensor read dry
278)
Gas to Pur Cols
203
3.0
0
No
Yes
Bubble contents in vessel
279)
End Loop
274
0.0
0
No
Yes
End water wash loop
280)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
281)
Gas to Pur Cols
203
8.0
0
No
Yes
Bubble water/ACN in vessels
282)
Return Sub
288
0.0
0
No
Yes
End of subroutine 8
283)
Sub Label
289
0.0
9
No
Yes
TFA detritylation/scrub and drain
284)
Set Pres Reg 3
303
0.0
4
No
Yes
Set regulator 3 to 4 psi
Cycles, Procedures, and System Messages C-29
Table C-6 Standard Purification Cycle (v4.52) (continued)
STEP
NUM
TIME
MIS
SNS
SAFE
285)
PurLowBlk Flush
FUNCTION
218
2.0
0
No
Yes
Flush lower purification block
286)
TFA to Pur Cols
188
6.0
0
No
Yes
Start split delivery of TFA to columns
287)
TFA to Pur Cols
188
25.0
0
Yes
Yes
Finish split delivery of TFA to sensors
288)
Wait
259
15.0
0
No
Yes
Initial detritylation/scrub wait
289)
Begin Loop
273
0.0
2
No
Yes
Begin detritylaylon/scrub loop
290)
TFA to PurCol A
189
1.0
999
No
Yes
Push fresh TFA over column A or wait
291)
TFA to PurCol B
190
1.0
999
No
Yes
Push fresh TFA over column B or wait
292)
TFA to PurCol C
191
1.0
999
No
Yes
Push fresh TFA over column C or wait
293)
Wait
259
15.0
0
No
Yes
Wait for detritylation
294)
End Loop
274
0.0
0
No
Yes
End detritylayion/scrub loop
295)
Begin Loop
273
0.0
6
No
Yes
Begin detritylaylon/scrub loop
296)
Gas to PurCol A
204
0.6
0
No
Yes
Gas push of fresh TFA over column A
297)
Gas to PurCol B
205
0.6
0
No
Yes
Gas push of fresh TFA over column B
298)
Gas to PurCol C
206
0.6
0
No
Yes
Gas push of fresh TFA over column C
299)
Pur Vent
220
2.0
0
No
Yes
Vent to allow TFA push
300)
Wait
259
23.0
0
No
Yes
Wait for detritylation
301)
End Loop
274
0.0
0
No
Yes
End detritylaylon/scrub loop
302)
Set Pres Reg 3
303
0.0
10
No
Yes
Set regulator 3 to 10 psi
303)
Tggle Vlves On
262
0.0
70
No
Yes
Select lower pur block to halog waste
304)
Back Flsh Pur A
208
12.0
0
No
Yes
Drain vessel A to halo waste
305)
Back Flsh Pur B
209
12.0
0
No
Yes
Drain vessel B to halo waste
306)
Back Flsh Pur C
210
12.0
0
No
Yes
Drain vessel C to halo waste
307)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
308)
Back Flsh Pur A
208
10.0
0
No
Yes
Dry line/column/vessel A
309)
Back Flsh Pur B
209
10.0
0
No
Yes
Dry line/column/vessel B
310)
Back Flsh Pur C
210
10.0
0
No
Yes
Dry line/column/vessel C
311)
Gas to Pur Cols
203
2.0
0
No
Yes
Parallel drv in reverse direction
312)
Back Flush Purs
207
8.0
0
No
Yes
Finish parallel dry line/column/vessel
313)
Tggle Vlves Off
263
0.0
70
No
Yes
Deselect lower pur block to halog waste
314)
Return Sub
288
0.0
0
No
Yes
End of subroutine 9
315)
Sub Label
289
0.0
10
No
Yes
Clean lower purification block
316)
H2O to PurWaste
187
1.0
0
No
Yes
Water rinse of lower pur block
317)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
318)
PurLowBlk Flush
218
6.0
0
No
Yes
Rinse lower pur block
319)
Return Sub
288
0.0
0
No
Yes
End of subroutine 10
320)
Sub Label
289
0.0
11
No
Yes
Set ramping function parameters
321)
Set Rmp Fxn Sns
265
0.0
10
No
Yes
Set ramp parms: # of total readings
322)
Set Rmp Fxn Trp
266
0.0
5
No
Yes
Set ramp parms: # of readings to trip
323)
Set Rmp Fxn Chk
267
0.0
5
No
Yes
Set ramp parms: # of readings to verify
324)
Set Rmp Fxn Dly
268
0.0
60
No
Yes
Set ramp parms: # of ms to delay verify
325)
Return Sub
288
0.0
0
No
Yes
End of subroutine 11
C-30 Cycles, Procedures, and System Messages
Comments
Table C-6 Standard Purification Cycle (v4.52) (continued)
STEP
NUM
TIME
MIS
SNS
SAFE
326)
Sub Label
FUNCTION
289
0.0
12
No
Yes
Wash UV cell and SC delivery line
Comments
327)
Set Pres Reg 3
303
0.0
10
No
Yes
Set regulator 3 to 10 psi
328)
SC Waste
242
0.0
0
No
Yes
Send SC to waste position
329)
H2O to PurWaste
187
3.0
0
No
Yes
Water rinse of lower pur block
330)
H2O to UV
214
3.0
0
No
Yes
Water rinse of UV cell
331)
PurLowBlk Flush
218
5.0
0
No
Yes
Flush lower pur block
332)
SC Block Flush
222
5.0
0
No
Yes
Flush lower pur blocks and UV cell dry
333)
Flsh UV toWaste
217
8.0
0
No
Yes
Flush UV cell dry
334)
Set Pres Reg 3
303
0.0
5
No
Yes
Set regulator 3 to 5 psi
335)
Flsh UV toWaste
217
3.0
0
No
Yes
Flush UV cell dry
336)
Return Sub
288
0.0
0
No
Yes
End of subroutine 12
337)
Sub Label
289
0.0
13
No
Yes
Not used
338)
Return Sub
288
0.0
0
No
Yes
End of subroutine 13
339)
Sub Label
289
0.0
14
No
Yes
Deliver ACN/water to vessels
340)
ACN to Pur Cols
173
4.0
0
No
Yes
Start split delivery of ACN to vessels
341)
ACN to Pur Cols
173
15.0
0
Yes
Yes
Finish split delivery of ACN to sensors
342)
H2O to Pur Cols
183
10.0
0
No
Yes
Parallel delivery of water to vessels
343)
Set Pres Reg 3
303
0.0
12
No
Yes
Set regulator 3 to 12 psi
344)
PurLowBlk Flush
218
5.0
0
No
Yes
Flush lower pur block
345)
Set Pres Reg 3
303
0.0
6
No
Yes
Set regulator 3 to 6 psi
346)
PurLowBlk Flush
218
2.0
0
No
Yes
Flush lower pur block
347)
Return Sub
288
0.0
0
No
Yes
End of subroutine 14
348)
Sub Label
289
0.0
15
No
Yes
Quantitate & collect for sample A
349)
UV Reading
216
2.0
1
No
Yes
Take UV reading (misc of 1 = A)
350)
Xfer UV to SC
215
15.0
0
No
Yes
Transfer oligos to SC
351)
Return Sub
288
0.0
0
No
Yes
End of subroutine 15
352)
Sub Label
289
0.0
16
No
Yes
Quantitate & collect for Sample B
353)
UV Reading
216
2.0
2
No
Yes
Take UV reading (misc of 2 = B)
354)
Xfer UV to SC
215
15.0
0
No
Yes
Transfer oligos to SC
355)
Return Sub
288
0.0
0
No
Yes
End of subroutine 16
356)
Sub Label
289
0.0
17
No
Yes
Quantitate & collect for sample C
357)
UV Reading
216
2.0
3
No
Yes
Take UV reading (misc of 3 = C)
358)
Xfer UV to SC
215
15.0
0
No
Yes
Transfer oligos to SC
359)
Return Sub
288
0.0
0
No
Yes
End of subroutine 17
360)
End of Cycle
272
0.0
0
No
Yes
Marks end of cycle and all subroutines
Cycles, Procedures, and System Messages C-31
Procedures
General Procedures are similar to cycles in that they consist of a set of functions that perform
an operation. Procedures, if performed during a run, are typically performed once,
while cycles are repeatedly performed during a run.
Edit Begin The ABI 3948 System is provided with one Begin procedure. The Start Up procedure
Procedure View primes all reagent delivery lines, from the reservoir to the reagent valve block, with
fresh reagent.
Always use this procedure prior to beginning a synthesis. Failure to
perform the Begin procedure can cause a synthesis failure.
IMPORTANT
The Start Up Procedure
An example of this procedure is listed in Table C-7. To modify the procedure, copy it
into a blank Begin procedure in the Edit Begin Procedure view (see “Edit Begin
Procedure View” on page 6-20).
Table C-7 The Start Up Procedure (v1.35 shown)
STEP
NUM
TIME
MISC
SNS
SAFE
1)
Begin of Cycle
FUNCTION
271
0.0
0
No
Yes
2)
Send Message
269
0.0
5
No
Yes
3)
DB 4.20H, 2.20H
110
0.0
0
No
Yes
4)
Clv Owns Depro
286
0.0
0
No
Yes
2)
Send Message
269
0.0
5
No
Yes
3)
DB 4.20H, 2.20H
110
0.0
0
No
Yes
4)
Clv Owns Depro
286
0.0
0
No
Yes
5)
Check Snsr Cal
320
0.0
0
No
Yes
6)
Heater Off
258
0.0
0
No
Yes
7)
Pres Reg AllOff
315
0.0
0
No
Yes
8)
Pres Reg AllOn
314
0.0
0
No
Yes
9)
Set Pres Reg 1
301
0.0
12
No
Yes
10)
Set Pres Reg 2
302
0.0
8
No
Yes
11)
Set Pres Reg 3
303
0.0
12
No
Yes
12)
Set Pres Reg 4
304
0.0
7
No
Yes
13)
Set Pres Reg 5
305
0.0
8
No
Yes
14)
Set Pres Reg 6
306
0.0
6
No
Yes
15)
Set Pres Reg 7
307
0.0
12
No
Yes
16)
Set Pres Reg 8
308
0.0
9
No
Yes
17)
Set Pres Reg 9
309
0.0
6
No
Yes
18)
Set Pres Reg 10
310
0.0
12
No
Yes
19)
Pres Reg AllOn
314
0.0
0
No
Yes
20)
Wait
259
15.0
0
No
Yes
21)
SynLowBlk Flush
65
5.0
0
No
Yes
22)
Go Sub
287
0.0
1
No
Yes
23)
IODINE to Waste
37
2.0
0
No
Yes
C-32 Cycles, Procedures, and System Messages
Table C-7 The Start Up Procedure (v1.35 shown) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
24)
Go Sub
FUNCTION
287
0.0
1
No
Yes
25)
TCA to Trityl
42
4.0
0
No
Yes
26)
ACN to Trityl
108
2.0
0
No
Yes
27)
LowTrityl Flush
106
5.0
0
No
Yes
28)
AC2O to Waste
31
2.0
0
No
Yes
29)
Go Sub
287
0.0
1
No
Yes
30)
NMI to Waste
32
2.0
0
No
Yes
31)
Go Sub
287
0.0
1
No
Yes
32)
T AMIDITE Waste
77
3.0
0
No
Yes
33)
C AMIDITE Waste
76
3.0
0
No
Yes
34)
G AMIDITE Waste
75
3.0
0
No
Yes
35)
A AMIDITE Waste
74
3.0
0
No
Yes
36)
8 AMIDITE Waste
81
3.0
0
No
Yes
37)
7 AMIDITE Waste
80
3.0
0
No
Yes
38)
6 AMIDITE Waste
79
3.0
0
No
Yes
39)
5 AMIDITE Waste
78
3.0
0
No
Yes
40)
Go Sub
287
0.0
1
No
Yes
41)
TET to Waste
52
2.0
0
No
Yes
42)
Go Sub
287
0.0
1
No
Yes
43)
Clv Blk Flush
144
10.0
0
No
Yes
44)
H2O to ClvWaste
130
5.0
0
No
Yes
45)
Clv Blk Flush
144
10.0
0
No
Yes
46)
Ammonia toWaste
115
3.0
0
No
Yes
47)
Dep Xfer Rinse
151
3.0
0
No
Yes
48)
Clv Blk Flush
144
10.0
0
No
Yes
49)
ACN to ClvWaste
120
2.0
0
No
Yes
50)
DepLowBlk Flush
153
5.0
0
No
Yes
51)
Gas to Coil A
156
15.0
0
No
Yes
52)
Gas to Coil B
157
15.0
0
No
Yes
53)
Gas to Coil C
158
15.0
0
No
Yes
54)
Pur Xfer Rinse
171
3.0
0
No
Yes
55)
Pur Xfer Flush
172
10.0
0
No
Yes
56)
Go Sub
287
0.0
2
No
Yes
57)
20%ACN to Waste
197
2.0
0
No
Yes
58)
Go Sub
287
0.0
2
No
Yes
59)
H2O to PurWaste
187
2.0
0
No
Yes
60)
Go Sub
287
0.0
2
No
Yes
61)
AcAcid to Waste
202
3.0
0
No
Yes
62)
Go Sub
287
0.0
2
No
Yes
63)
TFA to Waste
192
2.0
0
No
Yes
64)
Tggle Vlves On
262
0.0
70
No
Yes
Cycles, Procedures, and System Messages C-33
Table C-7 The Start Up Procedure (v1.35 shown) (continued)
STEP
NUM
TIME
MISC
SNS
SAFE
65)
PurLowBlk Flush
FUNCTION
218
2.0
0
No
Yes
66)
ACN to PurWaste
177
2.0
0
No
Yes
67)
PurLowBlk Flush
218
5.0
0
No
Yes
68)
Tggle Vlves Off
263
0.0
70
No
Yes
69)
TEAA to Waste
182
5.0
0
No
Yes
70)
PurLowBlk Flush
218
5.0
0
No
Yes
71)
ACN to PurWaste
177
5.0
0
No
Yes
72)
PurLowBlk Flush
218
5.0
0
No
Yes
73)
Go Sub
287
0.0
2
No
Yes
74)
SC Waste
242
0.0
0
No
Yes
75)
H2O to UV
214
10.0
0
No
Yes
76)
Flsh UV toWaste
217
10.0
0
No
Yes
77)
Xfer UV to SC
215
5.0
0
No
Yes
78)
SC Home
243
0.0
0
No
Yes
79)
SC Open
245
0.0
0
No
Yes
80)
Goto End
290
0.0
0
No
Yes
81)
Sub Label
289
0.0
1
No
Yes
82)
ACN to SynWaste
47
3.0
0
No
Yes
83)
SynLowBlk Flush
65
6.0
0
No
Yes
84)
Return Sub
288
0.0
0
No
Yes
85)
Sub Label
289
0.0
2
No
Yes
86)
ACN to PurWaste
177
3.0
0
No
Yes
87)
PurLowBlk Flush
218
5.0
0
No
Yes
88)
Return Sub
288
0.0
0
No
Yes
89)
End of Cycle
272
0.0
0
No
Yes
Edit End Procedure Use the End procedure to flush instrument reagent lines and valve blocks at the end of
View each synthesis run. An example of the standard end procedure provided with the
instrument is listed in Table C-8. To modify this procedure, copy it into a blank End
procedure (see “Edit End Procedure View” on page 6-21).
Table C-8
STEP
End Procedure (v1.11 shown)
FUNCTION
NUM
TIME
MISC
SNS
SAFE
1
Begin of Cycle
271
0.0
0
No
Yes
2
Heater Off
258
0.0
0
No
Yes
3
Set Pres Reg 1
301
0.0
12
No
Yes
4
Set Pres Reg 3
303
0.0
12
No
Yes
5
PurUprBlk Flush
219
20.0
0
No
Yes
6
PurLowBlk Flush
218
20.0
0
No
Yes
7
Tggle Vlves On
262
0.0
70
No
Yes
8
PurLowBlk Flush
218
20.0
0
No
Yes
9
Tggle Vlves Off
263
0.0
70
No
Yes
C-34 Cycles, Procedures, and System Messages
Table C-8
STEP
End Procedure (v1.11 shown) (continued)
NUM
TIME
MISC
SNS
SAFE
10
DepUprBlk Flush
FUNCTION
221
20.0
0
No
Yes
11
Set Pres Reg 10
310
0.0
12
No
Yes
12
DepLowBlk Flush
153
20.0
0
No
Yes
13
Clv Blk Flush
144
20.0
0
No
Yes
14
Set Pres Reg 7
307
0.0
12
No
Yes
15
Set Pres Reg 9
309
0.0
0
No
Yes
16
AMIDITE Vent
101
10.0
0
No
Yes
17
ACN to SynWaste
47
10.0
0
No
Yes
18
SynLowBlk Flush
65
20.0
0
No
Yes
19
LowTrityl Flush
106
20.0
0
No
Yes
20
Mix AMIDITE T
93
3.0
0
No
Yes
21
Mix AMIDITE C
92
3.0
0
No
Yes
22
Mix AMIDITE G
91
3.0
0
No
Yes
23
Mix AMIDITE A
90
3.0
0
No
Yes
24
Mix AMIDITE 8
97
3.0
0
No
Yes
25
Mix AMIDITE 7
96
3.0
0
No
Yes
26
Mix AMIDITE 6
95
3.0
0
No
Yes
27
Mix AMIDITE 5
94
3.0
0
No
Yes
28
Set Pres Reg 9
309
0.0
6
No
Yes
29
SynUprBlk Flush
105
20.0
0
No
Yes
30
Set Pres Reg 7
307
0.0
6
No
Yes
31
Set Pres Reg 10
310
0.0
12
No
Yes
32
Set Pres Reg 1
301
0.0
12
No
Yes
33
Dep Xfer Flush
152
5.0
0
No
Yes
34
Dep Xfer Rinse
151
3.0
0
No
Yes
35
Dep Xfer Flush
152
5.0
0
No
Yes
36
Gas to Coil A
156
15.0
0
No
Yes
37
Gas to Coil B
157
15.0
0
No
Yes
38
Gas to Coil C
158
15.0
0
No
Yes
39
H20 to Coil A
163
8.0
0
No
Yes
40
H20 to Coil B
164
8.0
0
No
Yes
41
H20 to Coil C
165
8.0
0
No
Yes
42
Gas to Coil A
156
15.0
0
No
Yes
43
Gas to Coil B
157
15.0
0
No
Yes
44
Gas to Coil C
158
15.0
0
No
Yes
45
Pur Xfer Flush
172
10.0
0
No
Yes
46
Pur Xfer Rinse
171
5.0
0
No
Yes
47
Pur Xfer Flush
172
10.0
0
No
Yes
48
Open All Jaws
237
0.0
0
No
Yes
49
Send Message
269
0.0
5
No
Yes
50
End of Cycle
272
0.0
0
No
Yes
Cycles, Procedures, and System Messages C-35
Edit Bottle Two types of procedures are located on the Edit Bottle Procedures view: Autodilute
Procedures View and Change procedures.
Phosphoramidite Autodilution
Use one of the Autodilute procedures to automatically dilute powdered β-cyanoethyl
phosphoramidites in 2.0 gram bottles. The table below lists the Autodilute procedures
available and the purpose of each.
IMPORTANT
During this procedure, back-flushed phosphoramidite and excess acetonitrile
contaminate the bottle contents. If you want to remove and save the phosphoramidites for later
use, do not initiate this procedure.
Table C-9 List of Phosphoramidite Autodilution Procedures
Name
Purpose
Autodil AGCT x.xx
Simultaneously autodilutes
phosphoramidites at Positions A,
G, C, and T.
Autodilute-A x.xx
Autodilutes phosphoramidite at
Position A.
Autodilute-G x.xx
Autodilutes phosphoramidite at
Position G.
Autodilute-C x.xx
Autodilutes phosphoramidite at
Position C.
Autodilute-T x.xx
Autodilutes phosphoramidite at
Position T.
Bottle Change Procedures (Manual Dilution)
Change Base (Chg base) Procedures for Positions A through G and Positions 5
through 8 on the instrument are intended for phosphoramidites manually diluted by the
user. After you have manually diluted a bottle of phosphoramidite, use the change
procedure for the phosphoramidite position on which you want to install the new bottle.
The change procedure provided in Table C-10 is representative of the Chg procedures
provided. Besides change procedures for the four phosphoramidite positions,
procedures are provided for changing tetrazole and ammonia.
Table C-10 Bottle Change Procedure
Step Function Name
Number
Time
Misc
SNS
Safe
271
0.0
0
No
Yes
1
Begin of Cycle
2
Set Press Reg 9
309
0.0
0
No
Yes
3
AMIDITE Vent
101
30.0
0
No
Yes
4
ACN AMIDITE 5
86
4.0
0
No
Yes
5
Mix ACN AMIDITE 5
94
5.0
0
No
Yes
6
Send Message
292
0.0
10
No
Yes
7
Send Message
292
0.0
1
No
Yes
8
Pause Chemistry
291
0.0
0
No
Yes
9
Set Press Reg 9
309
0.0
6
No
Yes
47
3.0
0
No
Yes
10 ACN to SynWaste
C-36 Cycles, Procedures, and System Messages
Table C-10 Bottle Change Procedure (continued)
Step Function Name
Number
Time
Misc
SNS
Safe
11 SynLowBlk Flush
65
3.0
0
No
Yes
12 End of Cycle
272
0.0
0
No
Yes
Miscellaneous The Miscellaneous Procedures view of the 3948Control software application contains
Procedures the following:
♦
A procedure for setting system pressures
♦
Procedures for calibrating the sensors and verifying calibration
♦
A procedure for priming amidite positions
♦
Procedures for cleaning columns and lines
♦
A procedure for emptying ACN lines
♦
Procedures for testing hardware
♦
Procedures for testing the pressure module and sample collector
♦
A Janitor procedure to clean up the system before shutdown
Cycles, Procedures, and System Messages C-37
System Messages
Instrument Status Instrument Status Messages
and Message The following instrument status messages appear in the upper right corner of all
Indicators Synthesizer Window views to indicate the current instrument condition:
♦
Ready - the instrument is idle and ready for a run to be initiated
♦
Running - the instrument is currently running chemistry
♦
Interrupted - a run in progress has been interrupted
♦
Manual Control - a manual control action is in progress
Note
It is important to be aware of what state the instrument is in at the time of a system
message.
Message Waiting/Critical Message Waiting
These messages are presented in the upper left corner of all Synthesizer Window
views except the Monitor Chemistry View, to indicate that an important message is
currently presented in the log on the bottom of the Monitor Chemistry View.
Types of Messages There are three types of messages generated for the 3948 instrument and flagged by
message indicators:
♦
Regular Reports to the Monitor Chemistry view
♦
Critical messages presented in the Monitor Chemistry window
♦
Microphone Only messages or messages stored in a Microphone file (not shown
in Monitor Chemistry).
These message types are flagged as follows:
♦
Regular Reports messages are flagged on any ABI 3948 System Control view by
“Message Waiting”.
♦
Critical messages are flagged on any ABI 3948 System Control view by “Critical
Message Waiting.”
♦
Microphone Only messages are not available during a run but can be examined at
the conclusion of the run.
Proceed to the log on the bottom of the Monitor Chemistry view to see the particular
message indicated by a message indicator.
C-38 Cycles, Procedures, and System Messages
Categories of Messages presented in the Monitor Chemistry view or in a Microphone file can be
Messages further categorized as Operator, Instrument, or General Activity messages:
♦
Operator messages are the messages prefaced by “Error:” or “Caution:”
♦
Instrument messages are the messages prefaced by “Attention:” or “Note”.
♦
General Activity messages are the messages without the “Error”, “Caution”,
“Attention”, or “Note” prefaces.
Some examples of conditions which may generate these three categories of
messages are listed under the discussions for the message types below. For a listing
of the relative severity of Operator and Instrument message, see “Hierarchical
Messages” on page C-43.
Message The type of activities which may be flagged by the three categories of message are
Conventions listed below:
Operator Messages
Operator messages, those messages prefaced by “Error” or “Caution,” are generated
by the types of activities outlined below.
Message Preface
Type of Activity
Error
Characterized by Cycle programming or Manual Control parameter
errors:
–
Using functions from the wrong chemistry module controller
–
Ramping function parameter(s) not set or out of range
–
Other out-of-range values (illegal pressure or regulator
number)
Note
Caution
Messages report values substituted at range limits.
Characterized by Discretionary bending of optimal operational
rules:
–
Allowing deliveries to open jaws
–
Setting temperature wait time too short to reach setpoint
Note
A hierarchy of Operator messages is presented under “Hierarchical Messages” on
page C-43.
Cycles, Procedures, and System Messages C-39
Instrument Messages
Instrument messages, those messages prefaced by “Attention” or “Note,” are
generated by the types of activities outlined below.
Message Preface
Type of Activity
Attention
Reports events likely to impact instrument performance
Note
–
- Transfers missed
–
- Sensor never triggered
–
- Jaw leak test failures and subsequent oligo drop outs
Indicates minor and/or infrequent operational events:
–
Retries
–
Recoveries
–
Sensor did not verify
–
Other miscellaneous events
General Activity Messages
General Activity messages, messages with no preface, are generated by the types of
activities outlined below.
Message Preface
Type of Activity
No leader or
message preface
Reports instrument actions and hardware status, mostly to
Microphone:
–
Run and Manual Control start/end/abort/pause/resume
–
Turntable moves
–
Jaw activity: open/close/leak test results
–
Regulator setpoint changes
–
Other: low pressure, a/d reference voltage, s/c motor
problems
–
Etc.
The three types of messages are listed in separate subsections below with the
information needed to interpret them.
Note
The examples provided in the three listings are representative of those you might see
in the Monitor Chemistry view window or in the Microphone file for a run and are generally self
explanatory.
Regular Reports This type of message contains all three categories of messages: Operator,
Messages Instrument, and General Activity.
Messages from these functions: Send Message (269) and Comment (270)
Note
The Send Message command is used in bottle change procedures to instruct the
operator to take some action.
Resetting the Database.
Pausing system.
Resuming System.
Pausing system: will auto-resume in 15 minutes.
Data Base has been reset.
C-40 Cycles, Procedures, and System Messages
Changed Instrument Name.
Pause Switch Pushed.
Resume with columns 4-6 in the load position.
Starting Chemistry Run: [Image version], [Database version].
Aborting Chemistry: [Image version], [Database version].
Chemistry Done: [Image version], [Database version].
Stopping Manual Control Action.
Aborting Manual Control.
Turntable move 10 completed.
Pausing System at turntable position 12.
Jaw pressure check retry failed during synthesis.
Initial synthesis pressure check failed: now retrying.
Regulator '23' does not exist.
Power Fail at 10/27/97 09:23:36.
A to D reference voltage 3.5 out of range.
Rack selection changed.
Note: turntable move retried.
Note: low input pressure detected.
Note: used Xfer Clv Col A recovery.
Note: function BackFlsh Purs retried 4 times.(to µphone only if <4)
Note: function TCA to Syn Cols retried A column 4 times.
Note: heater was turned off after 180 minutes.
Attention: Xfer Clv Col C transfer missed.
Attention: C+TET to column B sensor never triggered.
Attention: sensor never triggered at pur step 188.
Attention: leak detected in synthesis jaw/blocks.
Attention: oligos 10-12 discontinued during synthesis.
Caution: temp set to 35°, reached 36° in 300 seconds.
Error: cannot set regulator 10 to 15 psi, 12 psi limit used.
Error: used minimum heater setpoint of 15 instead of 10.
Error: used maximum heater setpoint of 70 instead of 85.
Error 0 readings requested: minimum valule of 1 used.
Error: 10 trip readings requested, used current max. 7.
Error: 10 check readings requested, used current max. 5.
Error: 20 readings requested: maximum value of 10 used.
Error: 10 trip readings reduced to new maximum of 7.s
Error: 10 check readings reduced to new maximum of 7.
Error: 20000ms sensor delay requested: maximum 2280.
Error: misc parameter must be: 1 = syn, 2 = clv, or 3 = pur.
Error: If First Cycle only works in synthesis cycles.
Error: End Row SCP/123 only works in Manual Control.
Error: no active oligos in cleavage to be discontinued.
Error: cannot end row because purification is inactive.
Error: oligos 7-9 now in cleavage are already discontinued.
Error: 999 heater time requested, used maximum 480.
Cycles, Procedures, and System Messages C-41
Critical Messages This type of message contains only the General Activity category of message.
Note
use.
Send Message function messages maybe be made 'critical' by selecting liquid sensor
Manual Control Paused: Resume instead of Running.
Sample Collector door open. Pausing System.
Jaw move failure. Pausing System.
Jaws open? Check jaws and Instrument Monitor Window.
Invalid RunS file: no cycles selected to run.
Invalid SeqL file: maximum sequence length exceeded.
Turntable May Be Out Of Position.
Regulators shut down due to low input pressure.
Synthesis jaw did not open.
Cleavage jaw did not close.
Sensor Calibration Problem. Pausing System.
Illegal valve set! Valves left off. Pausing System.
TTextFile::WriteData index = 501, max = 500
TTextFile::ReadData index = 501, max = 500
TStructFile::WriteData index = 501, max = 500
TStructFile::ReadData index = 501, max = 500
Jaws open: clv & pur! Valves left off. Pausing System.
Powerfail interrupted jaw open. Pausing System.
Powerfail interrupted jaw close. Pausing System.
Powerfail interrupted needle up move Pausing System.
Powerfail interrupted needle down move Pausing System.
Powerfail interrupted turntable move Pausing System.
Coil temperature 100°C is out of range. Pausing System.
Sample Collector Z Motor Timed Out. Pausing System.
Instrument in use: rack selection not changed.
Row ended: advance collector into position for next row.
Caution: reagent deliveries to open jaws enabled.
Error: Amidite bottle 'A' appears to be empty.
Microphone-only This type of message contains only the General Activity category of message.
messages
Starting Manual Control Action.
Pausing Manual Control.
Resuming Manual Control.
Manual Control Action Complete.
Coil temperature has been set to 35°C.
Coil temperature has been set to 65°C for deprotection.
Temp set to 38°, reached 38° in 281 seconds.
Name or Msg;Snsr; msecs; °C;Step; Ttbl(Microphone log column headings)
Synthesis A cycle ended.
Cleavage cycle ended.
Purification cycle ended.
Changing regulator 10 from 6 psi to 12 psi.
LogSensor Output (the standard sensor function report line)
Wet(11B) ≥ 9/10 Hg: 2232 1 1 1 1 1 1 1 1 1 5
14|14 (histogram)
Voltage[A]: -2.13
Results[A]: 3.2 odu 18.3 pmole
C-42 Cycles, Procedures, and System Messages
Syn P 0.01 N -.-- Clv P 0.02 N-.-- Pur F 0.54 P 0.49 (1st line of 2)
Tested at 12 psi, 2.00 passes
(2nd line of 2)
Ramping function parameter not set. Pausing System.
PSIs: 10.200 6.532 10.200 6.532 10.200 6.532 10.200 6.532 10.200 6.532
Leak Test: 10.02 9.98 0.04 P
Wait On Pur Flg; 00:12:21
Depro Heater/Fan Test
STARTING
Depro Heater Test
RUNNING Temp= 27, HtrCur=1200
Depro Fan Test
RUNNING Temp=35, FanCur= 60
Depro Heater/Fan Test
COMPLETE
Depro Htr Wait ensuring 60 minute deprotection; 00:12:21
Time Stamp; 00:12:21; 33
Coils have been hot for 63 minutes.
Synthesis jaw open elapsed time = 1567 ms.
Cleavage jaw close elapsed time = 1786 ms.
Sample Collector Z Motor Home Flag Error.
Note: function BackFlsh Purs retried 1 times.
Note: sensor 23B did not verify.
Note: function TCA to Syn Cols retried A column 3 times.
Hierarchical The list below presents a hierarchical list of Operator and Instrument category
Messages messages, ranging from conditions most likely to negatively affect a run at the top to
those least likely at the bottom. This hierarchy applies both to the individual messages
within each type (Error, Caution, Attention, Note) and to the relative severity of each of
these types of message.
Error: Amidite bottle 'G' appears to be empty.B+TET
Error: 0 readings requested, used minimum value 1.Ramping
Error: 20 readings requested, used maximum value 10.
Error: 10 trip readings reduced to new maximum of 7.
Error: 9 check readings reduced to new maximum of 7.
Error: 0 trip readings requested, used minimum value 1.
Error: 10 trip readings requested, used current max. 7.
Error: 9 check readings requested, used current max. 7.
Error: 20000ms sensor delay requested, used maximum 2280.
Error: used minimum heater setpoint of 15 instead of 10.Heater
Error: used maximum heater setpoint of 70 instead of 85.
Error: cannot set regulator 10 to 15 psi, 12 psi limit used.Other
Error: misc parameter must be: 1 = syn, 2 = clv, or 3 = pur.
Error: If First Cycle only works in synthesis cycles.
Error: End Row SCP/123 only works in Manual Control.
Error: no active oligos in cleavage to be discontinued.
Error: oligos 7-9 now in cleavage are already discontinued.
Error: 999 heater time requested, used maximum 480.
Caution: reagent deliveries to open jaws enabled.
Caution: temp set to 35°, reached 36° in 300 secs.
Attention: 'G'+TET to column B sensor never triggered.
Attention: sensor never triggered at pur step 10.
Attention: Xfer Clv Col C transfer missed.
Attention: Leak detected during in synthesis jaw/blocks.
Attention: Oligos 10-12 discontinued during synthesis.
Cycles, Procedures, and System Messages C-43
Note: function BackFlsh Purs retried 1 times.
Note: function TCA to Syn Cols retried A column 3 times.
Note: sensor 23B did not verify.
Note: used Xfer Clv Col A recovery.
Note: turntable move retried.
Note: low input pressure detected.
Note: heater was turned off after 180 minutes.
C-44 Cycles, Procedures, and System Messages
3948 System Function
List and Other Cycles D
D
In This Appendix
Topics Covered This appendix contains a detailed ABI 3948 System function list as well as the
additional purification cycles listed below:
Topic
See page
ABI 3848 System Function List
D-2
The Purification Dye Cycle
D-33
The Purification Biotin Cycle
D-42
ABI 3948 System Function List and Other Cycles D-1
ABI 3948 System Function List
List of Functions
Table D-1 lists the functions available for the ABI 3948 System.
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
1
Name
B+ TET to Syns
Valve List
4
22
Valve Description
Tetrazole
SynUpr to Waste
Note
Function 1 is a “macro” function. This means that the particular valves
used vary depending upon the sequence composition and the column to which
delivery is made. This function makes three separate serial amidite deliveries to
one column rather than a single simultaneous (parallel) delivery to all three
columns
2
3
4
5
6
7
A + TET to Syn A
G+ TET to Syn A
C+ TET to Syn A
T+ TET to Syn A
5+ TET to Syn A
6+ TET to Syn A
D-2 ABI 3948 System Function List and Other Cycles
4
Tetrazole
9
"A" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
10
"G" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
11
"C" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
12
"T" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
5
"5" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
6
"6" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
8
9
10
11
12
13
Name
7+ TET to Syn A
8+ TET to Syn A
A+ TET to Syn B
G+ TET to Syn B
C+ TET to Syn B
T+ TET to Syn B
Valve List
Tetrazole
7
"7" Amidite
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
8
"8" Amidit
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
9
"A" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
15
16
5+ TET to Syn B
6+ TET to Syn B
7+ TET to Syn B
Tetrazole
10
"G" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
11
"C" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
12
14
Valve Description
4
Tetrazole
"T" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
5
"5" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
6
"6" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
ABI 3948 System Function List and Other Cycles D-3
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
7
17
18
19
20
21
22
23
24
8+ TET to Syn B
A+ TET to Syn C
G+ TET to SynC
C+ TET to Syn C
T+ TET to Syn C
5+ TET to Syn C
6+ TET to Syn C
7+ TET to Syn C
D-4 ABI 3948 System Function List and Other Cycles
Valve Description
"7" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
8
"8" Amidite
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
9
"A" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
10
"G" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
11
"C" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
12
"T" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
5
"5" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
6
"6" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
7
"7" Amidite
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
25
Name
8+ TET to Syn C
26
Coupling Wait
27
CAP to Syn Cols
28
29
30
CAP to SynCol A
CAP to SynCol B
CAP to SynCol C
31
AC20 to Waste
32
NMI to Waste
Valve List
Valve Description
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
8
"8" Amidite
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
Waits according to the time in the B+TET
Calibration table.
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfr to SynLow
17
NMI
18
Ac20
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
15
Syn Col A Lower
16
Xfr to SynLow
17
NMI
18
Ac20
22
SynUpr to Waste
26
Syn Col A Upper
14
Syn Col B Lower
16
Xfr to SynLow
17
NMI
18
Ac20
22
SynUpr to Waste
25
Syn Col B Upper
13
Syn Col C Lower
16
Xfr to SynLow
17
NMI
18
Ac20
22
SynUpr to Waste
24
Syn Col C Upper
18
Ac20
21
SynLow to Waste
17
NMI
ABI 3948 System Function List and Other Cycles D-5
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
33
34
35
36
37
38
39
Name
IODINE to Syns
IODINE to Syn A
IODINE to Syn B
IODINE to Syn C
IODINE to Waste
TCA to Syn Cols
TCA to SynCol A
D-6 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
21
SynLow to Waste
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfr to SynLow
20
Iodine
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
15
Syn Col A Lower
16
Xfr to SynLow
20
Iodine
22
SynUpr to Waste
26
Syn Col A Upper
14
Syn Col B Lower
16
Xfr to SynLow
20
Iodine
22
SynUpr to Waste
25
Syn Col B Upper
13
Syn Col C Lower
16
Xfr to SynLow
20
Iodine
22
SynUpr to Waste
24
Syn Col C Upper
20
Iodine
21
SynLow to Waste
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfr to SynLow
19
TCA
23
SynUpr to Trityl
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
15
Syn Col A Lower
16
Xfr to SynLow
19
TCA
23
SynUpr to Trityl
26
Syn Col A Upper
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
40
41
42
43
44
Name
TCA to SynCol B
TCA to SynCol C
TCA to Trityl
ACN to Syn Cols
ACN to SynCol A
Valve List
Valve Description
14
Syn Col B Lower
16
Xfr to SynLow
19
TCA
23
SynUpr to Trityl
25
Syn Col B Upper
13
Syn Col C Lower
16
Xfr to SynLow
19
TCA
23
SynUpr to Trityl
24
Syn Col C Upper
19
TCA
21
SynLow to Waste
72
SynLow to Trityl
1
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
1
15
45
ACN to SynCol B
ACN to SynCol C
48
CN to SynWaste
TET to Syn Cols
Syn Col A Lower
SynUpr to Waste
26
Syn Col A Upper
1
ACN to Syn
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
1
13
47
ACN to Syn
22
14
46
ACN to Syn
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
1
ACN to Syn
16
Xfr to SynLow
21
SynLow to Waste
4
Tetrazole
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
22
SynUpr to Waste
ABI 3948 System Function List and Other Cycles D-7
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
49
50
51
52
53
54
55
56
57
Name
TET to SynCol A
TET to SynCol B
TET to Syn Col C
TET to Waste
Flush Syn Cols
Flush Syn Col A
Flush Syn Col B
Flush Syn Col C
Back Flush Syns
D-8 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
4
Tetrazole
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
4
Tetrazole
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
16
Xfr to SynLow
21
SynLow to Waste
2
Gas to Syn
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
2
Gas to Syn
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
2
Gas to Syn
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
2
Gas to Syn
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
58
59
60
61
62
63
64
Name
Back Flush Syn A
Back Flush Syn B
Back Flush Syn C
TritylFlsh Syns
TritylFlsh SynA
TritylFlsh SynB
TritylFlsh SynC
Valve List
Valve Description
16
Xfr to SynLow
21
SynLow to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
28
Gas to Syn Upper
15
Syn Col A Lower
16
Xfr to SynLow
21
SynLow to Waste
26
Syn Col A Upper
28
Gas to Syn Upper
14
Syn Col B Lower
16
Xfr to SynLow
21
SynLow to Waste
25
Syn Col B Upper
28
Gas to Syn Upper
13
Syn Col C Lower
16
Xfr to SynLow
21
SynLow to Waste
24
Syn Col C Upper
28
Gas to Syn Upper
2
Gas to Syn
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
23
SynUpr to Trityl
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
2
Gas to Syn
15
Syn Col A Lower
23
SynUpr to Trityl
26
Syn Col A Upper
2
Gas to Syn
14
Syn Col B Lower
23
SynUpr to Trityl
25
Syn Col B Upper
2
Gas to Syn
13
Syn Col C Lower
23
SynUpr to Trityl
24
Syn Col C Upper
ABI 3948 System Function List and Other Cycles D-9
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
65
66
67
68
Name
SynLowBlk Flush
Syn Upper Vent
Syn Lower Vent
ACN to AGCT
69
ACN to TET
70
ACN to TCA
71
72
73
74
75
ACN to IODINE
ACN to AC20
ACN to NMI
A AMIDITE Waste
G AMIDITE Waste
D-10 ABI 3948 System Function List and Other Cycles
Valve List
2
Valve Description
Gas to Syn
16
Xfr to SynLow
21
SynLow to Waste
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfr to SynLow
21
SynLow to Waste
1
ACN to Syn
9
“A” Amidite
10
“G” Amidite
11
“C” Amidite
12
“T” Amidite
76
Amidite Vent
1
ACN to Syn
4
Tetrazole
1
ACN to Syn
16
Xfr to SynLow
19
TCA
1
ACN to Syn
16
Xfr to SynLow
20
Iodine
1
ACN to Syn
16
Xfr to SynLow
18
Ac20
1
ACN to Syn
16
Xfr to SynLow
17
NMI
9
"A" Amidite
16
Xfr to SynLow
21
SynLow to Waste
10
"G" Amidite
16
Xfr to SynLow
21
SynLow to Waste
D 3948 Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
76
77
78
79
80
81
82
Name
C AMIDITE Waste
T AMIDITE Waste
5 AMIDITE Waste
6 AMIDITE Waste
7 AMIDITE Waste
8 AMIDITE Waste
ACN AMIDITE A
Valve List
11
"C" Amidite
16
Xfr to SynLow
21
SynLow to Waste
12
"T" Amidite
16
Xfr to SynLow
21
SynLow to Waste
5
84
85
86
ACN AMIDITE G
ACN AMIDITE C
ACN AMIDITE T
ACN AMIDITE 5
Xfr to SynLow
21
SynLow to Waste
6
88
89
ACN AMIDITE 6
ACN AMIDITE 7
ACN AMIDITE 8
"6" Amidite
16
Xfr to SynLow
21
SynLow to Waste
7
"7" Amidite
16
Xfr to SynLow
21
SynLow to Waste
8
"8" Amidite
16
Xfr to SynLow
21
SynLow to Waste
1
ACN to Syn
9
"A" Amidite
Amidite Vent
1
ACN to Syn
10
"G" Amidite
76
Amidite Vent
1
ACN to Syn
11
"C" Amidite
76
Amidite Vent
1
ACN to Syn
12
"T" Amidite
76
Amidite Vent
1
ACN to Syn
5
"5" Amidite
76
87
"5" Amidite
16
76
83
Valve Description
Amidite Vent
1
ACN to Syn
6
"6" Amidite
76
Amidite Vent
1
ACN to Syn
7
"7" Amidite
76
Amidite Vent
1
ACN to Syn
ABI 3948 System Function List and Other Cycles D-11
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
8
76
90
Mix AMIDITE A
92
93
94
Mix AMIDITE G
Mix AMIDITE C
Mix AMIDITE T
Mix AMIDITE 5
Gas to Syn
9
"A" Amidite
Gas to Syn
10
"G" Amidite
76
Amidite Vent
2
Gas to Syn
11
"C" Amidite
76
Amidite Vent
2
Gas to Syn
12
"T" Amidite
76
Amidite Vent
2
5
Mix AMIDITE 6
Mix AMIDITE 7
Mix AMIDITE 8
6
"6" Amidite
99
100
101
Mix AGCT
MIX AG
MIX CT
AMIDITE Vent
D-12 ABI 3948 System Function List and Other Cycles
Amidite Vent
2
Gas to Syn
7
"7" Amidite
Amidite Vent
2
Gas to Syn
8
8" Amidite
76
98
"5" Amidite
Amidite Vent
Gas to Syn
76
97
Gas to Syn
2
76
96
Amidite Vent
2
76
95
Amidite Vent
2
76
91
Valve Description
"8" Amidite
Amidite Vent
2
Gas to Syn
9
'A" Amidite
10
"G" Amidite
11
"C" Amidite
12
"T" Amidite
76
Amidite Vent
2
Gas to Syn
9
"A" Amidite
10
"G" Amidite
76
Amidite Vent
2
Gas to Syn
11
"C" Amidite
12
"T" Amidite
76
Amidite Vent
76
Amidite Vent
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
Valve Description
102
Ammonia Vent
74
Ammonia Vent
103
Mix Ammonia
29
Gas to Clv Lower
32
Ammonia
74
Ammonia Vent
104
Mix Tetrazole
105
SynUprBlk Flush
106
LowTrityl Flush
107
108
109
Numb
UprTrityl Flush
ACN to Trityl
4
Tetrazole
22
SynUpr to Waste
28
Gas to Syn Upper
2
Gas to Syn
16
Xfr to SynLow
21
SynLow to Waste
72
SynLow to Trityl
23
SynUpr to Trityl
28
Gas to Syn Upper
1
ACN to Syn
16
Xfer to SynLow
21
SynLow to Waste
72
SynLow to Trityl
Name
Database Version
111
Ammonia to Clvs
112
Ammonia to ClvA
113
Ammonia to ClvB
114
Ammonia to ClvC
115
Ammonia toWaste
117
Gas to Syn
Waste to Trityl
110
116
2
ACN to Clv Cols
ACN to ClvCol A
Purpose of Function
This function is used to indicate the
database version.
32
Ammonia
33
Clv Col C Lower
34
Clv Col B Lower
35
Clv Col A Lower
32
Ammonia
35
Clv Col A Lower
32
Ammonia
34
Clv Col B Lower
32
Ammonia
33
Clv Col C Lower
32
Ammonia
39
Xfr Clv to Dep
43
DepLow to Waste
33
Clv Col C Lower
34
Clv Col B Lower
35
Clv Col A Lower
38
ACN to Dep Lower
39
Xfr Clv to Dep
35
Clv Col A Lower
ABI 3948 System Function List and Other Cycles D-13
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
118
119
120
121
Name
ACN to ClvCol B
ACN to ClvCol C
ACN to ClvWaste
Gas to Clv Cols
122
Gas to ClvCol A
123
Gas to ClvCol B
124
Gas to ClvCol C
125
Gas to Clv II
126
127
128
129
130
131
H2O to Clv Cols
H2O to ClvCol A
H2O to ClvCol B
H2O to ClvCol C
H2O to ClvWaste
Back Flush Clvs
D-14 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
38
ACN to Dep Lower
39
Xfr Clv to Dep
34
Clv Col B Lower
38
ACN to Dep Lower
39
Xfr Clv to Dep
33
Clv Col C Lower
38
ACN to Dep Lower
39
Xfr Clv to Dep
38
ACN to Dep Lower
43
DepLow to Waste
29
Gas to Clv Lower
33
Clv Col C Lower
34
Clv Col B Lower
35
Clv Col A Lower
29
Gas to Clv Lower
35
Clv Col A Lower
29
Gas to Clv Lower
34
Clv Col B Lower
29
Gas to Clv Lower
33
Clv Col C Lower
33
Clv Col C Lower
34
Clv Col B Lower
35
Clv Col A Lower
37
Gas to Dep Lower
39
Xfr Clv to Dep
31
H2O to Clv Lower
33
Clv Col C Lower
34
Clv Col B Lower
35
Clv Col A Lower
31
H2O to Clv Lower
35
Clv Col A Lower
31
H2O to Clv Lower
34
Clv Col B Lower
31
H2O to Clv Lower
33
Clv Col C Lower
31
H2O to Clv Lower
39
Xfr Clv to Dep
43
DepLow to Waste
33
Clv Col C Lower
34
Clv Col B Lower
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
132
133
134
135
136
137
Name
Back flush Clv A
Back flush Clv B
Back flush Clv C
Crude A to UV
Crude B to UV
Crude C to UV
Valve List
Valve Description
35
Clv Col A Lower
39
Xfr Clv to Dep
43
DepLow to Waste
75
Gas to Clv Upper
35
Clv Col A Lower
39
Xfr Clv to Dep
43
DepLow to Waste
75
Gas to Clv Upper
34
Clv Col B Lower
39
Xfr Clv to Dep
43
DepLow to Waste
75
Gas to Clv Upper
33
Clv Col C Lower
39
Xfr Clv to Dep
43
DepLow to Waste
75
Gas to Clv Upper
37
Gas to Dep Lower
40
Coil A Input
46
Coil A Exit
49
Xfr from Dep
58
Xfr from Dep
62
Xfr to UV
37
Gas to Dep Lower
41
Coil B Input
47
Coil B Exit
49
Xfr from Dep
58
Xfr from Dep
62
Xfr to UV
37
Gas to Dep Lower
42
Coil C Input
48
Coil C Exit
49
Xfr from Dep
58
Xfr from Dep
62
Xfr to UV
29
Gas to Clv Lower
138
Press Line
139
Compress
75
Gas to Clv Upper
140
Clv Vent
33
Clv Col C Lower
34
Clv Col B Lower
35
Clv Col A Lower
39
Xfr Clv to Dep
43
DepLow to Waste
ABI 3948 System Function List and Other Cycles D-15
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
141
Name
Xfer Clv Col A
Valve List
Valve Description
35
Clv Col A Lower
39
Xfr Clv to Dep
40
Coil A Input
46
Coil A Exit
50
DepUpr to Waste
75
Gas to Clv Upper
Note
Functions 141, 142, and 143 “ramp up” the pressure from the regulator
until the sample leaves the coil during a cleavage cycle. In examining the cycle
in the Edit Cleavage Cycle view, the value in the MISC field is the regulator
being ramped and theTIME field is the time between ramps. These functions will
ramp up to four times before quitting. Ramping only works when “Yes” is
selected for the SNS field.
To observe the range of pressure over which one of these functions is ramped
for a particular regulator, change to the Monitor Instrument view while the
function is being executed.
The following functions are also ramping functions:
♦
135–137 Crude to UV
♦
162-168 Xfer Coil
♦
211-213 Pur to UV
142
143
144
Xfer Clv Col B
Xfer Clv Col C
Clv Blk Flush
D-16 ABI 3948 System Function List and Other Cycles
34
Clv Col B Lower
39
Xfr Clv to Dep
41
Coil B Input
47
Coil B Exit
50
DepUpr to Waste
75
Gas to Clv Upper
33
Clv Col C Lower
39
Xfr Clv to Dep
42
Coil C Input
48
Coil C Exit
50
DepUpr to Waste
75
Gas to Clv Upper
29
Gas to Clv Lower
39
Xfr Clv to Dep
43
DepLow to Waste
D 3948 Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
145
146
147
148
149
Name
Ammonia to SynA
Ammonia to SynB
Ammonia to SynC
Syn ACN to Clv
Flsh Syn to Clv
150
*User Fxn 150 *
151
Dep Xfer Rinse
152
153
154
155
Dep Xfer Flush
DepLowBlk Flush
DepUprBlk Rinse
Gas to Dep Coils
Valve List
3
Valve Description
Xfer from Syn
15
Syn Col A Lower
22
SynUpr to Waste
26
Syn Col A Upper
30
Xfr to Clv Lower
32
Ammonia
3
Xfer from Syn
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
30
Xfr to Clv Lower
32
Ammonia
3
Xfer from Syn
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
30
Xfr to Clv Lower
32
Ammonia
1
ACN to Syn
3
Xfer from Syn
30
Xfr to Clv Lower
39
Xfer Clv to Dep
43
DepLow to Waste
2
Gas to Syn
3
Xfer from Syn
30
Xfr to Clv Lower
39
Xfer Clv to Dep
43
DepLow to Waste
31
H2O to Clv Lower
39
Xfr Clv to Dep
43
DepLow to Waste
29
Gas to Clv Lower
39
Xfr Clv to Dep
43
DepLow to Waste
37
Gas to Dep Lower
43
DepLow to Waste
45
H2O to Dep Upper
50
DepUpr to Waste
37
Gas to Dep Lower
ABI 3948 System Function List and Other Cycles D-17
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
156
157
158
159
160
161
162
163
Name
Gas to Coil A
Gas to Coil B
Gas to Coil C
ACN to Coil A
ACN to Coil B
ACN to Coil C
H2O toDep Coils
H2O to Coil A
D-18 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
40
Coil A Input
41
Coil B Input
42
Coil C Input
46
Coil A Exit
47
Coil B Exit
48
Coil C Exit
50
DepUpr to Waste
37
Gas to Dep Lower
40
Coil A Input
46
Coil A Exit
50
DepUpr to Waste
37
Gas to Dep Lower
41
Coil B Input
47
Coil B Exit
50
DepUpr to Waste
37
Gas to Dep Lower
42
Coil C Input
48
Coil C Exit
50
DepUpr to Waste
38
ACN to Dep Lower
40
Coil A Input
46
Coil A Exit
50
DepUpr to Waste
38
ACN to Dep Lower
41
Coil B Input
47
Coil B Exit
50
DepUpr to Waste
38
ACN to Dep Lower
42
Coil C Input
48
Coil C Exit
50
DepUpr to Waste
40
Coil A Input
41
Coil B Input
42
Coil C Input
43
DepLow to Waste
45
H2O to Dep Upper
46
Coil A Exit
47
Coil B Exit
48
Coil C Exit
40
Coil A Input
43
DepLow to Waste
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
164
165
Name
H2O to Coil B
H2O to Coil C
Valve List
Valve Description
45
H2O to Dep Upper
46
Coil A Exit
41
Coil B Input
43
DepLow to Waste
45
H2O to Dep Upper
47
Coil B Exit
42
Coil C Input
43
DepLow to Waste
45
H2O to Dep Upper
48
Coil C Exit
Note
Functions 166, 167, and 168 “ramp up” the pressure from the regulator
until the sample is transferred during a purification cycle. In examining the cycle
in the Edit Purification Cycle view, the value in the MISC field is the regulator
being ramped.
To observe the range of pressure over which one of these functions is ramped
for a particular regulator, change to the Monitor Instrument view while the
function is being executed.
See material on page B-5 for more information.
166
167
168
Xfr Coil A
Xfr Coil B
Xfr Coil C
37
Gas to Dep Lower
40
Coil A Input
46
Coil A Exit
49
Xfr from Dep
58
Xfr from Dep
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
37
Gas to Dep Lower
41
Coil B Input
47
Coil B Exit
49
Xfr from Dep
58
Xfr from Dep
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waster
37
Gas to Dep Lower
42
Coil C Input
48
Coil C Exit
49
Xfr from Dep
58
Xfr from Dep
59
Pur Col C Lower
65
Pur Col C Upper
ABI 3948 System Function List and Other Cycles D-19
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
Valve Description
68
PurUpr to Waster
169
Depro Htr Wait
MISC field states how long the heater
must be hot (in whole minutes). If the
heater hasn’t been hot at least that
long, the time the function must wait (in
seconds) is calculated and the heater
stays hot for the calculated time.
170
Start Depro Htr
Starts the heater ramping up to the
temperature specified in the MISC field.
Deprotection time begins once the
heater reaches the specified
temperature.
171
Pur Xfer Rinse
172
173
174
175
176
Pur Xfer Flush
ACN to Pur Cols
ACN Pur Col A
ACN Pur Col B
ACN Pur Col C
45
H2O to Dep Upper
49
Xfr from Dep
58
Xfr from Dep
63
PurLow to Waste
44
Gas to Dep Upper
49
Xfr from Dep
58
Xfr from Dep
63
PurLow to Waste
52
ACN to Pur
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
52
ACN to Pur
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
52
ACN to Pur
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waste
52
ACN to Pur
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
Valve Description
Numb
Name
Valve List
177
ACN to PurWaste
52
ACN to Pur
63
Pur Low to Waste
D-20 ABI 3948 System Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
178
179
180
181
182
183
184
185
Numb
186
Name
TEAA to Pur Cols
TEAA to Pur A
TEAA to Pur B
TEAA to Pur C
TEAA to Waste
H2O to Pur Cols
H2O to Pur Col A
H2O to Pur Col B
Name
H2O to Pur Col C
Valve List
Valve Description
53
TEAA
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
53
TEAA
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
53
TEAA
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waste
53
TEAA
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
53
TEAA
63
Pur Low to Waste
56
H2O to Pur
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
56
H2O to Pur
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
56
H2O to Pur
60
Pur Col B Lower
66
Pur Col B Upper
Valve List
Valve Description
68
PurUpr to Waste
56
H2O to Pur
ABI 3948 System Function List and Other Cycles D-21
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
187
H2O to PurWaste
188
TFA to Pur Cols
189
190
191
192
193
194
Numb
TFA to PurCol A
TFA to PurCol B
TFA to PurCol C
TFA to Waste
20%ACN to Purs
20%ACN to Pur A
Name
D-22 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
56
H2O to Pur
63
Pur Low to Waste
54
TFA
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
54
TFA
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
54
TFA
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waste
54
TFA
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
54
TFA
63
Pur Low to Waste
57
20% ACN
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
57
20% ACN
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
Valve List
Valve Description
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
195
196
197
198
199
200
201
Name
20%ACN to Pur B
20%ACN to Pur C
20% ACN to Waste
AcAcid to Purs
AcAcid to Pur A
AcAcid to Pur B
AcAcid to Pur C
202
AcAcid to Waste
203
Gas to Pur Cols
204
Gas to PurCol A
Valve List
Valve Description
57
20% ACN
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waste
57
20% ACN
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
57
20% ACN
63
Pur Low to Waste
55
Acetic Acid
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
55
Acetic Acid
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
55
Acetic Acid
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waste
55
Acetic Acid
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
55
Acetic Acid
63
PurLow to Waste
51
Gas to Pur
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
68
PurUpr to Waste
51
Gas to Pur
ABI 3948 System Function List and Other Cycles D-23
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
205
206
207
208
209
210
211
212
Name
Gas to PurCol B
Gas to PurCol C
Back Flush Purs
Back Flsh Pur A
Back Flsh Pur B
Back Flsh Pur C
Pur A to UV
Pur B to UV
D-24 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
61
Pur Col A Lower
67
Pur Col A Upper
68
PurUpr to Waste
51
Gas to Pur
60
Pur Col B Lower
66
Pur Col B Upper
68
PurUpr to Waste
51
Gas to Pur
59
Pur Col C Lower
65
Pur Col C Upper
68
PurUpr to Waste
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
63
Pur Low to Waste
65
Pur Col C Upper
66
Pur Col B Upper
67
Pur Col A Upper
69
Gas to Pur Upper
61
Pur Col A Lower
63
Pur Low to Waste
67
Pur Col A Upper
69
Gas to Pur Upper
60
Pur Col B Lower
63
Pur Low to Waste
66
Pur Col B Upper
69
Gas to Pur Upper
59
Pur Col C Lower
63
Pur Low to Waste
65
Pur Col C Upper
69
Gas to Pur Upper
61
Pur Col A Lower
62
Xfr to UV
67
Pur Col A Upper
69
Gas to Pur Upper
60
Pur Col B Lower
62
Xfr to UV
66
Pur Col B Upper
69
Gas to Pur Upper
D 3948 Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
213
214
Name
Pur C to UV
H2O to UV
215
Xfer UV to SC
216
UV Reading
217
Flush UV toWaste
218
PurLowBlk Flush
219
PurUprBlk Flush
220
Pur Vent
221
DepUprBlk Flush
222
SC Block Flush
223
Set UV Scale
224
Pur Gas to Dep
225
226
Pur ACN to Dep
Pur H2O to Dep
227
*User Fxn 227 *
228
*User Fxn 228 *
Valve List
Valve Description
59
Pur Col C Lower
62
Xfr to UV
65
Pur Col C Upper
69
Gas to Pur Upper
56
H2O to Pur
62
Xfr to UV
77
UV Lower
78
Gas to UV
77
UV Lower
78
Gas to UV
79
UV to Waste
51
Gas to Pur
63
Pur Low to Waste
68
PurUpr to Waste
69
Gas to Pur Upper
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
63
Pur Low to Waste
44
Gas to Dep Upper
50
DepUpr to Waste
51
Gas to Pur
62
Xfr to UV
Adjusts ODU calculation with a scaling
factor expressed as a percent.
49
Xfer from Dep
50
DepUpr to Waste
51
Gas to Pur
58
Xfer from Dep
49
Xfer from Dep
50
DepUpr to Waste
52
ACN to Pur
58
Xfer from Dep
49
Xfer from Dep
50
DepUpr to Waste
56
H2O to Pur
58
Xfer from Dep
ABI 3948 System Function List and Other Cycles D-25
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
229
End Row SCP/123
230
Go Sub On Fail
231
Syn Jaw Open
232
Syn Jaw Close
233
Clv Jaw Open
234
Clv Jaw Close
235
Pur Jaw Open
236
Pur Jaw Close
2
Gas to Syn
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfer to SynLow
28
Gas to Syn Upper
29
Gas to Clv Lower
39
Xfer Clv to Dep
75
Gas to Clv Upper
49
Xfer from Dep
51
Gas to Pur
58
Xfer from Dep
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
69
Gas to Pur Upper
237
Open All Jaws
238
Close All Jaws
239
TurnTable Home
240
TurnTable Next
241
TurnTable Prev
242
SC Waste
Puts needle over sample vials.
243
SC Home
Sends tray to “home” position.
244
SC Next
Sends tray to the next position.
245
SC Open
Sends tray to the front.
246
SC Close
Sends tray to correct position according
to index.
247
SC Needle Up
248
SC Needle Down
249
Relay 1 On
250
Relay 1 Off
251
Relay 1 Pulse
252
Relay 2 On
253
Relay 2 Off
254
Relay 2 Pulse
255
Fan On
256
Fan Off
D-26 ABI 3948 System Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
257
Heater On
258
Heater Off
259
Wait
260
Set Coil Temp
261
Wait Coil Temp
Cycle waits for heater to reach temp. in
the Misc field, cycle resumes when
temp. is reached or when step times
out. On timeout, temp. reached before
going on is reported in the status
window (Monitor Chemistry).
262
Tggle Vlves On
Turns single valve ON until Tggle Vlves
Off function is used.
263
Tggle Vlves Off
Used to turn OFF single valves
activated by the Tggle Vlves On
function.
264
SC Prev
Sends tray to the previous position
according to index.
265
Set Rmp Fxn Sns
266
Set Rmp Fxn Trp
267
Set Rmp Fxn Chk
268
Set Rmp Fxn Dly
These functions are used to set
parameters which help define the
behavior of the ramping functions.
See 265 Set Rmp Fxn Sns, et al in
Appendix A.
269
Send Message
270
Comment
271
Begin of Cycle
272
End of Cycle
273
Begin Loop
274
End Loop
275
Start Here
“cycle entry” - First Synthesis cycle
skips to this step to begin.
276
Exit DMT On
This step executes only on the last
synthesis cycle of any given column.
See the discussion in Appendix A.
277
If Pur Col A
Execute steps to Else or Endif if Col A
is active, otherwise skips steps.
278
If Pur Col B
Execute steps to Else or Endif if Col B
is active, otherwise skips steps.
279
If Pur Col C
Execute steps to Else or Endif if Col C
is active, otherwise skips steps.
280
Else
Execute when previous If statement
condition is false;
281
Endif
End of If statement.
282
Select Pur Cols
Picks columns that require purification
(no Crude).
283
Select Act Cols
Picks active columns, crude or pure.
These two parameters are used to send
a message as specified in the MISC
field. See 269 Send Message/
270Comment in Appendix A.
These functions serve only to mark the
beginning and ending steps of a cycle
procedure.
These functions mark the beginning
and ending boundaries of a block of
steps to be executed repeatedly.
ABI 3948 System Function List and Other Cycles D-27
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
284
Clv Wants Depro
Cleavage module waits on Pur flag
reset.
285
Pure Owns Depro
(I) - sets flag at the beginning to “Pur.”
Flag indicates that purification “owns”
deprotection coils.
286
Clv Owns Depro
(O) - resets flag at the end to “Not Pur ”
- releases deprotection coils to
cleavage.
287
Go Sub
288
Return Sub
289
Sub Label
290
Goto End
291
If Any Act Cols
Executes steps to nearest Else or
Endif, only if there are columns
purifying - deactivates all non-purifying
columns.
292
Go Sub A
Do subroutine (Go Sub A) only if
column is active. Deactivates other
columns until Return Sub.
293
Go Sub B
Do subroutine (Go Sub B) only if
column is active. Deactivates other
columns until Return Sub.
294
Go Sub C
Do subroutine (Go Sub C) only if
column is active. Deactivates other
columns until Return Sub.
295
If First Cycle
296
If Last Cycle
297
If Crude Col A
298
If Crude Col B
299
If Crude Col C
300
Engage All Cols
301
Set Pres Reg 1
302
Set Pres Reg 2
303
Set Pres Reg 3
304
Set Pres Reg 4
305
Set Pres Reg 5
306
Set Pres Reg 6
307
Set Pres Reg 7
308
Set Pres Reg 8
309
Set Pres Reg 9
310
Set Pres Reg 10
311
Set Press RegAll
312
Press Reg On
313
Press Reg Off
314
Pres Reg AllOn
D-28 ABI 3948 System Function List and Other Cycles
D 3948 Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
Valve Description
315
Pres Reg AllOff
316
If Cyc Greater
317
Save Reg Pres
318
Restore RegPres
319
Revive Act Cols
320
Check Snsr Cal
Checks all sensor calibrations. “Wet
readings” are at least 2X “dry readings.”
321
CalibrateGas Sns
Takes a dry sensor calibration reading.
322
CalibrateLiqSns
Takes a wet sensor calibration reading.
323
Calib AllGasSns
324
Calib AllLiqSns
325
Jaw Test Time
326
Pres SynJaws
Used during instrument assembly.
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
28
Gas to Syn Upper
327
Pres ClvJaws
75
Gas to Clv Upper
328
Pres PurJaws
51
Gas to Pur
59
Pur Col C Lower
60
Pur Col B Lower
61
Pur Col A Lower
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfr to SynLow
17
NMI
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
329
Begin Leak Test
330
End Leak Test
331
NMI to Cols
332
333
NMI to Col A
NMI to Col B
15
Syn Col A Lower
16
Xfr to SynLow
17
NMI
22
SynUpr to Waste
26
Syn Col A Upper
14
Syn Col B Lower
16
Xfr to SynLow
17
NMI
22
SynUpr to Waste
ABI 3948 System Function List and Other Cycles D-29
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
334
335
336
337
338
339
340
Name
NMI to Col C
AC20 to Cols
AC20 to Col A
AC20 to Col B
AC20 to Col C
Tet to Test
Tet to Test A
D-30 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
25
Syn Col B Upper
13
Syn Col C Lower
16
Xfr to SynLow
17
NMI
22
SynUpr to Waste
24
Syn Col C Upper
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
16
Xfr to SynLow
18
Ac20
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
15
Syn Col A Lower
16
Xfr to SynLow
18
Ac20
22
SynUpr to Waste
26
Syn Col A Upper
14
Syn Col B Lower
16
Xfr to SynLow
18
Ac20
22
SynUpr to Waste
25
Syn Col B Upper
13
Syn Col C Lower
16
Xfr to SynLow
18
Ac20
22
SynUpr to Waste
24
Syn Col C Upper
4
Tetrazole
13
Syn Col C Lower
14
Syn Col B Lower
15
Syn Col A Lower
22
SynUpr to Waste
24
Syn Col C Upper
25
Syn Col B Upper
26
Syn Col A Upper
4
Tetrazole
15
Syn Col A Lower
22
SynUpr to Waste
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
341
342
343
344
345
346
347
348
349
350
Name
Tet to Test B
Tet to Test C
A to Col B
G to Col B
C to Col B
T to Col B
5 to Col B
6 to Col B
7 to Col B
8 to Col B
Valve List
Valve Description
26
Syn Col A Upper
4
Tetrazole
14
Syn Col B Lower
22
SynUpr to Waste
25
Syn Col B Upper
4
Tetrazole
13
Syn Col C Lower
22
SynUpr to Waste
24
Syn Col C Upper
9
"A" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
10
"G" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
11
"C" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
12
"T" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
5
"5" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
6
"6" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
7
"7" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
8
"8" Amidite
14
Syn Col B Lower
22
Syn Upr to Waste
25
Syn Col B Upper
ABI 3948 System Function List and Other Cycles D-31
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
351
352
353
354
355
356
357
Name
Fish SynA to SC
Flsh SynB to SC
Flsh SynC to SC
Flsh ClvA to SC
Flsh ClvB to SC
Flsh ClvC to SC
ACN to Ammonia
D-32 ABI 3948 System Function List and Other Cycles
Valve List
3
Valve Description
Xfr from Syn
15
Syn Col A Lower
26
Syn Col A Upper
28
Gas to Syn Upper
30
Xfr to Clv Lower
36
Mfg Test 36
55
Acetic Acid
62
Xfr to UV
3
Xfr from Syn
14
Syn Col B Lower
25
Syn Col B Upper
28
Gas to Syn Upper
30
Xfr to Clv Lower
36
Mfg Test 36
55
Acetic Acid
62
Xfr to UV
3
Xfr from Syn
13
Syn Col C Lower
24
Syn Col C Upper
28
Gas to Syn Upper
30
Xfr to Clv Lower
36
Mfg Test 36
55
Acetic Acid
62
Xfr to UV
35
Clv Col A Lower
55
Acetic Acid
62
Xfr to UV
75
Gas to Clv Upper
36
Mfg Test 36
34
Clv Col B Lower
55
Acetic Acid
62
Xfr to UV
75
Gas to Clv Upper
36
Mfg Test 36
33
Clv Col C Lower
55
Acetic Acid
62
Xfr to UVr
75
Gas to Clv Uppe
36
Mfg Test v36
32
Ammonia
38
ACN to Dep Lower
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
39
358
359
360
361
362
363
364
365
366
367
368
369
370
ACN to TEAA
ACN to TFA
ACN to H2O(1)
ACN to H2O(2)
ACN to H2O(3)
ACN to 20%ACN
ACN to V23
ACN to V70 NC
ACN to SC NC
ACN to V43
ACN to V70 NO
ACN to V79 NC
ACN to DEPRO A
Valve Description
Xfr Clv to Dep
52
ACN to Pur
53
TEAA
52
ACN to Pur
54
TFA
52
ACN to Pur
56
H2O to Pur
31
H2O to Clv Lower
38
ACN to Dep Lower
39
Xfr Clv to Dep
38
ACN to Dep Lower
40
Coil A Input
45
H2O to Dep Upper
46
Coil A Exit
52
ACN to Pur
57
20% ACN
1
ACN to Syn
13
Syn Col C Lower
23
SynUpr to Trityl
24
Syn Col C Upper
52
ACN to Pur
63
Pur Low to Waste
70
Xfr to Trityl
52
ACN to Pur
62
Xfr to UVr
1
ACN to Syn
3
Xfr from Syn
30
Xfr to Clv Lower
39
Xfr Clv to Dep
43
Dep Low to Waste
52
ACN to Pur
63
Pur Low to Waste
52
ACN to Pur
62
Xfr to UV
77
UV Lower
79
UV to Waste
38
ACN to Dep Lower
40
Coil A Input
46
Coil A Exit
50
Dep Upr to Waste
ABI 3948 System Function List and Other Cycles D-33
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
371
372
373
374
375
376
377
378
379
Name
ACN to DEPRO B
ACN to DEPRO C
R3-UV-SC
R3-UV-Waste
ACN to AcAcid
Depro Xfer SC
Flsh DepA to SC
Flsh DepB to SC
Flsh DepC to SC
D-34 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
38
ACN to Dep Lower
41
Coil B Input
47
Coil B Exit
50
Dep Upr to Waste
38
ACN to Dep Lower
42
Coil C Input
48
Coil C Exit
50
Dep Upr to Waste
51
Gas to Pur
62
Xfr to UV
77
UV Lower
78
Gas to UV
51
Gas to Pur
62
Xfr to UV
77
UV Lower
78
Gas to UV
79
UV to Waste
55
Acetic Acid
52
ACN to Pur
36
Mfg Test v36
37
Gas to De Lower
39
Xfr Clv to Dep
55
Acetic Acid
62
Xfr to UV
37
Gas to Dep Lower
40
Coil A Input
46
Coil A Exit
49
Xfr from Dep
58
Xfr from Dep
62
Xfr to UV
37
Gas to Dep Lower
41
Coil B Input
47
Coil B Exit
49
Xfr from Dep
58
Xfr from Dep
62
Xfr to UV
37
Gas to Dep Lower
42
Coil C lnput
48
Coil C Exit
49
Xfr from Dep
58
Xfr from Dep
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
Valve List
62
380
*User Fxn 380 *
381
*User Fxn 381 *
382
*User Fxn 382 *
383
*User Fxn 383 *
384
*User Fxn 384 *
385
*User Fxn 385 *
386
*User Fxn 386 *
387
*User Fxn 387 *
388
*User Fxn 388 *
389
*User Fxn 389 *
390
*User Fxn 390 *
391
*User Fxn 391 *
392
*User Fxn 392 *
393
*User Fxn 393 *
394
*User Fxn 394 *
395
Pause Chemistry
396
Time Stamp
397
Test Valves
398
Depro Test
399
If Test To Do
400
* Reserved *
401
SynUpr Wet 401
402
SynUprWet A 402
403
SynUprWet B 403
404
SynUprWet C 404
405
SynUpr Wet 405
406
SynUprWet A 406
407
SynUprWet B 407
408
SynUprWet C 408
409
SynUpr Wet 409
410
SynUprWet A 410
411
SynUprWet B 411
412
SynUprWet C 412
413
SynUpr Dry 413
414
SynUprDry A 414
415
SynUprDry B 415
416
SynUprDry C 416
Valve Description
Xfr to UV
Report current instrument time with
timestamp # to Microphone.
Execute cycle steps to Endif if Test is
selected (the test to perform is coded in
the Misc field.
ABI 3948 System Function List and Other Cycles D-35
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
417
SynMan Wet 417
418
SynManWet A 418
419
SynManWet B 419
420
SynManWet C 420
421
SynMan Wet 421
422
SynManWet A 422
423
SynManWet B 423
424
SynManWet C 424
425
SynMan Wet 425
426
SynManWet A 426
427
SynManWet B 427
428
SynManWet C 428
429
SynLow Wet 429
430
SynLowWet A 430
431
SynLowWet B 431
432
SynLowWet C 432
433
SynLow Dry 433
434
SynLowDry A 434
435
SynLowDry B 435
436
SynLowDry C 436
437
Clv Wet 437
438
Clv Wet A 438
439
Clv Wet B 439
440
Clv Wet C 440
441
Clv Wet 441
442
Clv Wet A 442
443
Clv Wet B 443
444
Clv Wet C 444
445
Clv Dry 445
446
Clv Dry A 446
447
Clv Dry B 447
448
Clv Dry C 448
449
Clv Dry 449
450
Clv Dry A 450
451
Clv Dry B 451
452
Clv Dry C 452
453
Coil Wet 453
454
Coil Wet A 454
455
Coil Wet B 455
D-36 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
D 3948 Function List and Other Cycles
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
456
Coil Wet C 456
457
Coil Dry 457
458
Coil Dry A 458
459
Coil Dry B 459
460
Coil Dry C 460
461
Pur Wet 461
462
Pur Wet A 462
463
Pur Wet B 463
464
Pur Wet C 464
465
Pur Wet 465
466
Pur Wet A 466
467
Pur Wet B 467
468
Pur Wet C 468
469
Pur Wet 469
470
Pur Wet A 470
471
Pur Wet B 471
472
Pur Wet C 472
473
Pur Wet 473
474
Pur Wet A 474
475
Pur Wet B 475
476
Pur Wet C 476
477
Pur Wet 477
478
Pur Wet A 478
479
Pur Wet B 479
480
Pur Wet C 480
481
Pur Dry 481
482
Pur Dry A 482
483
Pur Dry B 483
484
Pur Dry C 484
485
Pur Dry 485
486
Pur Dry A 486
487
Pur Dry B 487
488
Pur Dry C 488
489
UV Wet 489
490
UV Wet 490
491
UV Wet 491
492
UV Wet 492
493
UV Wet 493
494
UV Wet 494
495
UV Wet 495
Valve List
Valve Description
ABI 3948 System Function List and Other Cycles D-37
Table D-1 ABI 3948 System Function List (v4.20, 2.20)
Numb
Name
496
UV Wet 496
497
UV Wet 497
498
UV Dry 498
499
UV Dry 499
500
UV Dry 500
D-38 ABI 3948 System Function List and Other Cycles
Valve List
Valve Description
The Purification Dye Cycle
A
Listing of Dye Cycle
D 3948 Function List and Other Cycles
Table D-2 Pur v.4.40 Dye Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
1
Begin of Cycle
271
0.0
0
No
Yes
2
Pur Owns Depro
285
0.0
0
No
Yes
3
If Any Act Cols
291
0.0
0
No
Yes
4
Else
280
0.0
0
No
Yes
5
Wait
259
60.0
0
No
Yes
6
Wait
259
60.0
0
No
Yes
7
Wait
259
60.0
0
No
Yes
8
SC Next
244
0.0
0
No
Yes
9
SC Next
244
0.0
0
No
Yes
10
SC Next
244
0.0
0
No
Yes
11
SC Open
245
0.0
0
No
Yes
12
Wait Coil Temp
261
0.0
0
No
Yes
13
Set Coil Temp
260
0.0
0
No
Yes
14
Clv Owns Depro
286
0.0
0
No
Yes
15
Goto End
290
0.0
0
No
Yes
16
Endif
281
0.0
0
No
Yes
17
Pur Jaw Close
236
0.0
0
No
Yes
18
If Any Act Cols
291
0.0
0
No
Yes
19
Else
280
0.0
0
No
Yes
20
Revive Act Cols
319
0.0
0
No
Yes
21
Go Sub
287
0.0
11
No
Yes
22
Depro Htr Wait
169
0.0
0
No
Yes
23
Wait Coil Temp
261
0.0
0
No
Yes
24
Set Coil Temp
260
0.0
0
No
Yes
25
Go Sub
287
0.0
2
No
Yes
26
Go Sub
287
0.0
1
No
Yes
27
Go Sub
287
0.0
12
No
Yes
28
SC Close
246
0.0
0
No
Yes
29
SC Needle Down
248
0.0
0
No
Yes
30
Go Sub A
292
0.0
6
No
Yes
31
Xfer UV to SC
215
15.0
0
No
Yes
32
Go Sub
287
0.0
2
No
Yes
33
Go Sub
287
0.0
1
No
Yes
34
Go Sub
287
0.0
12
No
Yes
35
SC Next
244
0.0
0
No
Yes
36
Go Sub B
293
0.0
6
No
Yes
37
Xfer UV to SC
215
15.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-39
Table D-2 Pur v.4.40 Dye Cycle
STEP
NUM
TIME
MISC
SNS
SAFE
38
FUNCTION NAME
Go Sub
287
0.0
2
No
Yes
39
Go Sub
287
0.0
1
No
Yes
40
Go Sub
287
0.0
12
No
Yes
41
SC Next
244
0.0
0
No
Yes
42
Go Sub C
294
0.0
6
No
Yes
43
Xfer UV to SC
215
15.0
0
No
Yes
44
Go Sub
287
0.0
2
No
Yes
45
Go Sub
287
0.0
12
No
Yes
46
SC Next
244
0.0
0
No
Yes
47
SC Open
245
0.0
0
No
Yes
48
Clv Owns Depro
286
0.0
0
No
Yes
49
Goto End
290
0.0
0
No
Yes
50
Endif
281
0.0
0
No
Yes
51
Time Stamp
396
0.0
30
No
Yes
52
Set Pres Reg 3
303
0.0
12
No
Yes
53
PurLowBlk Flush
218
5.0
0
No
Yes
54
ACN to PurWaste
177
5.0
0
No
Yes
55
PurLowBlk Flush
218
5.0
0
No
Yes
56
Go Sub
287
0.0
3
No
Yes
57
Go Sub
287
0.0
8
No
Yes
58
Go Sub
287
0.0
3
No
Yes
59
Select Pur Cols
282
0.0
0
No
Yes
60
If Any Act Cols
291
0.0
0
No
Yes
61
Go Sub
287
0.0
5
No
Yes
62
Go Sub
287
0.0
3
No
Yes
63
Go Sub
287
0.0
5
No
Yes
64
Go Sub
287
0.0
3
No
Yes
65
Go Sub
287
0.0
4
No
Yes
66
Go Sub
287
0.0
3
No
Yes
67
Go Sub
287
0.0
4
No
Yes
68
Go Sub
287
0.0
3
No
Yes
69
Go Sub
287
0.0
9
No
Yes
70
Go Sub
287
0.0
9
No
Yes
71
Go Sub
287
0.0
5
No
Yes
72
Go Sub
287
0.0
3
No
Yes
73
Go Sub
287
0.0
5
No
Yes
74
Go Sub
287
0.0
3
No
Yes
75
Begin Loop
273
0.0
2
No
Yes
76
ACN to Pur Cols
173
2.2
0
No
Yes
77
ACN to Pur Cols
173
15.0
0
Yes
Yes
78
H2O to PurCol A
184
5.0
0
No
Yes
D-40 ABI 3948 System Function List and Other Cycles
Table D-2 Pur v.4.40 Dye Cycle
STEP
NUM
TIME
MISC
SNS
SAFE
79
FUNCTION NAME
H2O to PurCol B
185
5.0
0
No
Yes
80
H2O to PurCol C
186
5.0
0
No
Yes
81
Gas to Pur Cols
203
15.0
0
No
Yes
82
Go Sub
287
0.0
3
No
Yes
83
End Loop
274
0.0
0
No
Yes
84
Go Sub
287
0.0
4
No
Yes
85
Go Sub
287
0.0
3
No
Yes
86
Set Pres Reg 3
303
0.0
9
No
Yes
87
TEAA to Waste
182
5.0
0
No
Yes
88
TEAA to PurCols
178
25.0
0
No
Yes
89
TEAA to PurCols
178
45.0
0
Yes
Yes
90
PurUprBlk Flush
219
2.0
0
No
Yes
91
Gas to PurCol A
204
10.0
0
No
Yes
92
Gas to PurCol B
205
10.0
0
No
Yes
93
Gas to PurCol C
206
10.0
0
No
Yes
94
Set Pres Reg 3
303
0.0
12
No
Yes
95
Back Flsh Pur A
208
20.0
0
No
Yes
96
Back Flsh Pur B
209
20.0
0
No
Yes
97
Back Flsh Pur C
210
20.0
0
No
Yes
98
Begin Loop
273
0.0
3
No
Yes
99
Back Flsh Pur A
208
10.0
0
No
Yes
100
Back Flsh Pur B
209
10.0
0
No
Yes
101
Back Flsh Pur C
210
10.0
0
No
Yes
102
End Loop
274
0.0
0
No
Yes
103
Go Sub
287
0.0
10
No
Yes
104
Endif
281
0.0
0
No
Yes
105
Select Act Cols
283
0.0
0
No
Yes
106
Go Sub C
294
0.0
2
No
Yes
107
Go Sub
287
0.0
11
No
Yes
108
Time Stamp
396
0.0
31
No
Yes
109
Depro Htr Wait
169
0.0
0
No
Yes
110
Time Stamp
396
0.0
32
No
Yes
111
Wait Coil Temp
261
0.0
0
No
Yes
112
Set Coil Temp
260
0.0
0
No
Yes
113
Time Stamp
396
0.0
33
No
Yes
114
Xfer Coil C
168
12.0
1
Yes
No
115
Xfer Coil C
168
20.0
0
No
No
116
Go Sub B
293
0.0
2
No
Yes
117
Xfer Coil B
167
12.0
1
Yes
No
118
Xfer Coil B
167
20.0
0
No
No
119
Go Sub A
292
0.0
2
No
Yes
ABI 3948 System Function List and Other Cycles D-41
Table D-2 Pur v.4.40 Dye Cycle
STEP
NUM
TIME
MISC
SNS
SAFE
120
FUNCTION NAME
Xfer Coil A
166
12.0
1
Yes
No
121
Xfer Coil A
166
20.0
0
No
No
122
Time Stamp
396
0.0
34
No
Yes
123
Clv Owns Depro
286
0.0
0
No
Yes
124
Go Sub
287
0.0
10
No
Yes
125
Select Pur Cols
282
0.0
0
No
Yes
126
If Any Act Cols
291
0.0
0
No
Yes
127
Set Pres Reg 3
303
0.0
9
No
Yes
128
Gas to Pur Cols
203
60.0
0
No
Yes
129
Set Pres Reg 3
303
0.0
5
No
Yes
130
PurLowBlk Flush
218
2.0
0
No
Yes
131
Begin Loop
273
0.0
2
No
Yes
132
AcAcid to Waste
202
2.0
0
No
Yes
133
AcAcid to Purs
198
3.0
0
No
Yes
134
AcAcid to Purs
198
30.0
0
Yes
Yes
135
Gas to Pur Cols
203
25.0
0
No
Yes
136
ACN to PurWaste
177
2.0
0
No
Yes
137
PurLowBlk Flush
218
5.0
0
No
Yes
138
End Loop
274
0.0
0
No
Yes
139
Gas to Pur Cols
203
15.0
0
No
Yes
140
Go Sub A
292
0.0
7
No
Yes
141
Go Sub B
293
0.0
7
No
Yes
142
Go Sub C
294
0.0
7
No
Yes
143
Go Sub
287
0.0
4
No
No
144
Go Sub
287
0.0
3
No
Yes
145
Go Sub
287
0.0
4
No
Yes
146
Go Sub
287
0.0
3
No
Yes
147
Begin Loop
273
0.0
3
No
No
148
Go Sub
287
0.0
4
No
No
149
Go Sub
287
0.0
3
No
Yes
150
End Loop
274
0.0
0
No
No
151
20%ACN to Waste
197
4.0
0
No
Yes
152
20%ACN to Purs
193
6.0
0
No
Yes
153
20%ACN to Purs
193
15.0
0
Yes
Yes
154
Go Sub
287
0.0
10
No
Yes
155
Set Pres Reg 3
303
0.0
4
No
Yes
156
PurLowBlk Flush
218
2.0
0
No
Yes
157
Gas to Pur Cols
203
10.0
0
No
Yes
158
Endif
281
0.0
0
No
Yes
159
Time Stamp
396
0.0
35
No
Yes
160
Select Act Cols
283
0.0
0
No
Yes
D-42 ABI 3948 System Function List and Other Cycles
Table D-2 Pur v.4.40 Dye Cycle
STEP
NUM
TIME
MISC
SNS
SAFE
161
FUNCTION NAME
Go Sub A
292
0.0
1
No
Yes
162
Go Sub A
292
0.0
12
No
Yes
163
SC Close
246
0.0
0
No
Yes
164
SC Needle Down
248
0.0
0
No
Yes
165
Gas to PurCol A
204
7.0
0
No
Yes
166
Pur A to UV
211
10.0
3
Yes
Yes
167
Pur A to UV
211
20.0
0
No
Yes
168
Go Sub A
292
0.0
14
No
Yes
169
Go Sub A
292
0.0
15
No
Yes
170
Go Sub B
293
0.0
1
No
Yes
171
Go Sub B
293
0.0
12
No
Yes
172
SC Next
244
0.0
0
No
Yes
173
Gas to PurCol B
205
7.0
0
No
Yes
174
Pur B to UV
212
10.0
3
Yes
Yes
175
Pur B to UV
212
20.0
0
No
Yes
176
Go Sub B
293
0.0
14
No
Yes
177
Go Sub B
293
0.0
16
No
Yes
178
Go Sub C
294
0.0
1
No
Yes
179
Go Sub C
294
0.0
12
No
Yes
180
SC Next
244
0.0
0
No
Yes
181
Gas to PurCol C
206
7.0
0
No
Yes
182
Pur C to UV
213
10.0
3
Yes
Yes
183
Pur C to UV
213
20.0
0
No
Yes
184
Go Sub C
294
0.0
14
No
Yes
185
Go Sub C
294
0.0
17
No
Yes
186
Go Sub
287
0.0
12
No
Yes
187
SC Next
244
0.0
0
No
Yes
188
SC Open
245
0.0
0
No
Yes
189
Time Stamp
396
0.0
36
No
Yes
190
Set Pres Reg 3
303
0.0
5
No
Yes
191
Gas to Pur Cols
203
45.0
0
Yes
Yes
192
Gas to Pur Cols
203
10.0
0
No
Yes
193
Go Sub
287
0.0
3
No
Yes
194
Go Sub
287
0.0
8
No
Yes
195
Go Sub
287
0.0
3
No
Yes
196
Time Stamp
396
0.0
37
No
Yes
197
Goto End
290
0.0
0
No
Yes
198
Sub Label
289
0.0
1
No
Yes
199
Set Pres Reg 3
303
0.0
6
No
Yes
200
SC Waste
242
0.0
0
No
Yes
201
H2O to PurWaste
187
5.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-43
Table D-2 Pur v.4.40 Dye Cycle
STEP
NUM
TIME
MISC
SNS
SAFE
202
FUNCTION NAME
H2O to UV
214
5.0
0
No
Yes
203
SC Block Flush
222
5.0
0
No
Yes
204
Wait
259
10.0
0
No
Yes
205
UV Reading
216
2.0
0
No
Yes
206
Flsh UV toWaste
217
6.0
0
No
Yes
207
H2O to UV
214
1.0
0
No
Yes
208
SC Block Flush
222
10.0
0
No
Yes
209
Xfer UV to SC
215
10.0
0
No
Yes
210
Return Sub
288
0.0
0
No
Yes
211
Sub Label
289
0.0
2
No
Yes
212
Set Pres Reg 1
301
0.0
12
No
Yes
213
Set Pres Reg 3
303
0.0
12
No
Yes
214
ACN to PurWaste
177
0.5
0
No
Yes
215
PurLowBlk Flush
218
3.0
0
No
Yes
216
Pur Xfer Rinse
171
2.0
0
No
Yes
217
Pur Xfer Flush
172
15.0
0
No
Yes
218
Set Pres Reg 1
301
0.0
8
No
Yes
219
DepLowBlk Flush
153
2.0
0
No
No
220
Return Sub
288
0.0
0
No
No
221
Sub Label
289
0.0
3
No
Yes
222
Set Pres Reg 3
303
0.0
12
No
Yes
223
Back Flush Purs
207
6.0
0
No
Yes
224
Back Flush Purs
207
45.0
0
Yes
Yes
225
Back Flsh Pur A
208
6.0
0
No
Yes
226
Back Flsh Pur B
209
6.0
0
No
Yes
227
Back Flsh Pur C
210
6.0
0
No
Yes
228
Begin Loop
273
0.0
2
No
Yes
229
Gas to Pur Cols
203
1.0
0
No
Yes
230
Back Flush Purs
207
4.0
0
No
Yes
231
End Loop
274
0.0
0
No
Yes
232
Return Sub
288
0.0
0
No
Yes
233
Sub Label
289
0.0
4
No
Yes
234
Set Pres Reg 3
303
0.0
9
No
Yes
235
H2O to Pur Cols
183
3.0
0
No
Yes
236
H2O to Pur Cols
183
15.0
0
Yes
Yes
237
Gas to Pur Cols
203
15.0
0
Yes
Yes
238
Gas to Pur Cols
203
6.0
0
No
Yes
239
Return Sub
288
0.0
0
No
Yes
240
Sub Label
289
0.0
5
No
Yes
241
Set Pres Reg 3
303
0.0
4
No
Yes
242
ACN to Pur Cols
173
4.0
0
No
Yes
D-44 ABI 3948 System Function List and Other Cycles
Table D-2 Pur v.4.40 Dye Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
243
ACN to Pur Cols
173
15.0
0
Yes
Yes
244
Gas to Pur Cols
203
15.0
0
Yes
Yes
245
Gas to Pur Cols
203
6.0
0
No
Yes
246
Return Sub
288
0.0
0
No
Yes
247
Sub Label
289
0.0
6
No
Yes
248
Crude A to UV
135
12.0
1
Yes
Yes
249
Crude A to UV
135
20.0
0
No
Yes
250
Crude B to UV
136
12.0
1
Yes
Yes
251
Crude B to UV
136
20.0
0
No
Yes
252
Crude C to UV
137
12.0
1
Yes
Yes
253
Crude C to UV
137
20.0
0
No
Yes
254
Return Sub
288
0.0
0
No
Yes
255
Sub Label
289
0.0
7
No
Yes
256
Set Pres Reg 3
303
0.0
12
No
Yes
257
ACN to PurWaste
177
2.0
0
No
Yes
258
PurLowBlk Flush
218
5.0
0
No
Yes
259
Set Pres Reg 3
303
0.0
6
No
Yes
260
Back Flush Purs
207
15.0
0
No
Yes
261
Back Flush Purs
207
45.0
3
Yes
Yes
262
Set Pres Reg 3
303
0.0
12
No
Yes
263
Back Flush Purs
207
15.0
0
No
Yes
264
Return Sub
288
0.0
0
No
Yes
265
Sub Label
289
0.0
8
No
Yes
266
Set Pres Reg 3
303
0.0
6
No
Yes
267
ACN to Pur Cols
173
4.0
0
No
Yes
268
ACN to Pur Cols
173
15.0
0
Yes
Yes
269
Gas to Pur Cols
203
15.0
0
Yes
Yes
270
Gas to Pur Cols
203
3.0
0
No
Yes
271
Set Pres Reg 3
303
0.0
8
No
Yes
272
Begin Loop
273
0.0
2
No
Yes
273
H2O to Pur Cols
183
4.0
0
No
Yes
274
H2O to Pur Cols
183
15.0
0
Yes
Yes
275
Gas to Pur Cols
203
15.0
0
Yes
Yes
276
Gas to Pur Cols
203
3.0
0
No
Yes
277
End Loop
274
0.0
0
No
Yes
278
Set Pres Reg 3
303
0.0
12
No
Yes
279
Gas to Pur Cols
203
8.0
0
No
Yes
280
Return Sub
288
0.0
0
No
Yes
281
Sub Label
289
0.0
9
No
Yes
282
Set Pres Reg 3
303
0.0
4
No
Yes
283
PurLowBlk Flush
218
2.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-45
Table D-2 Pur v.4.40 Dye Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
284
TFA to Pur Cols
188
6.0
0
No
Yes
285
TFA to Pur Cols
188
25.0
0
Yes
Yes
286
Wait
259
15.0
0
No
Yes
287
Begin Loop
273
0.0
2
No
Yes
288
TFA to PurCol A
189
1.0
999
No
Yes
289
TFA to PurCol B
190
1.0
999
No
Yes
290
TFA to PurCol C
191
1.0
999
No
Yes
291
Wait
259
15.0
0
No
Yes
292
End Loop
274
0.0
0
No
Yes
293
Begin Loop
273
0.0
6
No
Yes
294
Gas to PurCol A
204
0.6
0
No
Yes
295
Gas to PurCol B
205
0.6
0
No
Yes
296
Gas to PurCol C
206
0.6
0
No
Yes
297
Pur Vent
220
2.0
0
No
Yes
298
Wait
259
23.0
0
No
Yes
299
End Loop
274
0.0
0
No
Yes
300
Set Pres Reg 3
303
0.0
10
No
Yes
301
Tggle Vlves On
262
0.0
70
No
Yes
302
Back Flsh Pur A
208
12.0
0
No
Yes
303
Back Flsh Pur B
209
12.0
0
No
Yes
304
Back Flsh Pur C
210
12.0
0
No
Yes
305
Set Pres Reg 3
303
0.0
12
No
Yes
306
Back Flsh Pur A
208
10.0
0
No
Yes
307
Back Flsh Pur B
209
10.0
0
No
Yes
308
Back Flsh Pur C
210
10.0
0
No
Yes
309
Gas to Pur Cols
203
2.0
0
No
Yes
310
Back Flush Purs
207
8.0
0
No
Yes
311
Tggle Vlves Off
263
0.0
70
No
Yes
312
Return Sub
288
0.0
0
No
Yes
313
Sub Label
289
0.0
10
No
Yes
314
H2O to PurWaste
187
1.0
0
No
Yes
315
Set Pres Reg 3
303
0.0
12
No
Yes
316
PurLowBlk Flush
218
6.0
0
No
Yes
317
Return Sub
288
0.0
0
No
Yes
318
Sub Label
289
0.0
11
No
Yes
319
Set Rmp Fxn Sns
265
0.0
10
No
Yes
320
Set Rmp Fxn Trp
266
0.0
5
No
Yes
321
Set Rmp Fxn Chk
267
0.0
5
No
Yes
322
Set Rmp Fxn Dly
268
0.0
60
No
Yes
323
Return Sub
288
0.0
0
No
Yes
324
Sub Label
289
0.0
12
No
Yes
D-46 ABI 3948 System Function List and Other Cycles
Table D-2 Pur v.4.40 Dye Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
325
Set Pres Reg 3
303
0.0
10
No
Yes
326
SC Waste
242
0.0
0
No
Yes
327
H2O to PurWaste
187
3.0
0
No
Yes
328
H2O to UV
214
3.0
0
No
Yes
329
PurLowBlk Flush
218
5.0
0
No
Yes
330
SC Block Flush
222
5.0
0
No
Yes
331
Flsh UV toWaste
217
8.0
0
No
Yes
332
Set Pres Reg 3
303
0.0
5
No
Yes
333
Flsh UV toWaste
217
3.0
0
No
Yes
334
Return Sub
288
0.0
0
No
Yes
335
Sub Label
289
0.0
13
No
Yes
336
Return Sub
288
0.0
0
No
Yes
337
Sub Label
289
0.0
14
No
Yes
338
ACN to Pur Cols
173
4.0
0
No
Yes
339
ACN to Pur Cols
173
15.0
0
Yes
Yes
340
H2O to Pur Cols
183
10.0
0
No
Yes
341
Set Pres Reg 3
303
0.0
12
No
Yes
342
PurLowBlk Flush
218
5.0
0
No
Yes
343
Set Pres Reg 3
303
0.0
6
No
Yes
344
PurLowBlk Flush
218
2.0
0
No
Yes
345
Return Sub
288
0.0
0
No
Yes
346
Sub Label
289
0.0
15
No
Yes
347
UV Reading
216
2.0
1
No
Yes
348
Xfer UV to SC
215
15.0
0
No
Yes
349
Return Sub
288
0.0
0
No
Yes
350
Sub Label
289
0.0
16
No
Yes
351
UV Reading
216
2.0
2
No
Yes
352
Xfer UV to SC
215
15.0
0
No
Yes
353
Return Sub
288
0.0
0
No
Yes
354
Sub Label
289
0.0
17
No
Yes
355
UV Reading
216
2.0
3
No
Yes
356
Xfer UV to SC
215
15.0
0
No
Yes
357
Return Sub
288
0.0
0
No
Yes
358
End of Cycle
272
0.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-47
The Purification Biotin Cycle
Listing of Biotin
Cycle
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
1
Begin of Cycle
271
0.0
0
No
Yes
2
Pur Owns Depro
285
0.0
0
No
Yes
3
If Any Act Cols
291
0.0
0
No
Yes
4
Else
280
0.0
0
No
Yes
5
Wait
259
60.0
0
No
Yes
6
Wait
259
60.0
0
No
Yes
7
Wait
259
60.0
0
No
Yes
8
SC Next
244
0.0
0
No
Yes
9
SC Next
244
0.0
0
No
Yes
10
SC Next
244
0.0
0
No
Yes
11
SC Open
245
0.0
0
No
Yes
12
Wait Coil Temp
261
0.0
0
No
Yes
13
Set Coil Temp
260
0.0
0
No
Yes
14
Clv Owns Depro
286
0.0
0
No
Yes
15
Goto End
290
0.0
0
No
Yes
16
Endif
281
0.0
0
No
Yes
17
Pur Jaw Close
236
0.0
0
No
Yes
18
If Any Act Cols
291
0.0
0
No
Yes
19
Else
280
0.0
0
No
Yes
20
Revive Act Cols
319
0.0
0
No
Yes
21
Go Sub
287
0.0
11
No
Yes
22
Depro Htr Wait
169
0.0
0
No
Yes
23
Wait Coil Temp
261
0.0
0
No
Yes
24
Set Coil Temp
260
0.0
0
No
Yes
25
Go Sub
287
0.0
2
No
Yes
26
Go Sub
287
0.0
1
No
Yes
27
Go Sub
287
0.0
12
No
Yes
28
SC Close
246
0.0
0
No
Yes
29
SC Needle Down
248
0.0
0
No
Yes
30
Go Sub A
292
0.0
6
No
Yes
31
Xfer UV to SC
215
15.0
0
No
Yes
32
Go Sub
287
0.0
2
No
Yes
33
Go Sub
287
0.0
1
No
Yes
34
Go Sub
287
0.0
12
No
Yes
35
SC Next
244
0.0
0
No
Yes
36
Go Sub B
293
0.0
6
No
Yes
37
Xfer UV to SC
215
15.0
0
No
Yes
D-48 ABI 3948 System Function List and Other Cycles
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
2
No
Yes
38
Go Sub
287
0.0
39
Go Sub
287
0.0
1
No
Yes
40
Go Sub
287
0.0
12
No
Yes
41
SC Next
244
0.0
0
No
Yes
42
Go Sub C
294
0.0
6
No
Yes
43
Xfer UV to SC
215
15.0
0
No
Yes
44
Go Sub
287
0.0
2
No
Yes
45
Go Sub
287
0.0
12
No
Yes
46
SC Next
244
0.0
0
No
Yes
47
SC Open
245
0.0
0
No
Yes
48
Clv Owns Depro
286
0.0
0
No
Yes
49
Goto End
290
0.0
0
No
Yes
50
Endif
281
0.0
0
No
Yes
51
Time Stamp
396
0.0
30
No
Yes
52
Set Pres Reg 3
303
0.0
12
No
Yes
53
PurLowBlk Flush
218
5.0
0
No
Yes
54
ACN to PurWaste
177
5.0
0
No
Yes
55
PurLowBlk Flush
218
5.0
0
No
Yes
56
Go Sub
287
0.0
3
No
Yes
57
Go Sub
287
0.0
8
No
Yes
58
Go Sub
287
0.0
3
No
Yes
59
Select Pur Cols
282
0.0
0
No
Yes
60
If Any Act Cols
291
0.0
0
No
Yes
61
Go Sub
287
0.0
5
No
Yes
62
Go Sub
287
0.0
3
No
Yes
63
Go Sub
287
0.0
5
No
Yes
64
Go Sub
287
0.0
3
No
Yes
65
Go Sub
287
0.0
4
No
Yes
66
Go Sub
287
0.0
3
No
Yes
67
Go Sub
287
0.0
4
No
Yes
68
Go Sub
287
0.0
3
No
Yes
69
Go Sub
287
0.0
9
No
Yes
70
Go Sub
287
0.0
9
No
Yes
71
Go Sub
287
0.0
5
No
Yes
72
Go Sub
287
0.0
3
No
Yes
73
Go Sub
287
0.0
5
No
Yes
74
Go Sub
287
0.0
3
No
Yes
75
Begin Loop
273
0.0
2
No
Yes
76
ACN to Pur Cols
173
2.2
0
No
Yes
77
ACN to Pur Cols
173
15.0
0
Yes
Yes
78
H2O to PurCol A
184
5.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-49
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
79
H2O to PurCol B
185
5.0
0
No
Yes
80
H2O to PurCol C
186
5.0
0
No
Yes
81
Gas to Pur Cols
203
15.0
0
No
Yes
82
Go Sub
287
0.0
3
No
Yes
83
End Loop
274
0.0
0
No
Yes
84
Go Sub
287
0.0
4
No
Yes
85
Go Sub
287
0.0
3
No
Yes
86
Set Pres Reg 3
303
0.0
9
No
Yes
87
TEAA to Waste
182
5.0
0
No
Yes
88
TEAA to PurCols
178
25.0
0
No
Yes
89
TEAA to PurCols
178
45.0
0
Yes
Yes
90
PurUprBlk Flush
219
2.0
0
No
Yes
91
Gas to PurCol A
204
10.0
0
No
Yes
92
Gas to PurCol B
205
10.0
0
No
Yes
93
Gas to PurCol C
206
10.0
0
No
Yes
94
Set Pres Reg 3
303
0.0
12
No
Yes
95
Back Flsh Pur A
208
20.0
0
No
Yes
96
Back Flsh Pur B
209
20.0
0
No
Yes
97
Back Flsh Pur C
210
20.0
0
No
Yes
98
Begin Loop
273
0.0
3
No
Yes
99
Back Flsh Pur A
208
10.0
0
No
Yes
100
Back Flsh Pur B
209
10.0
0
No
Yes
101
Back Flsh Pur C
210
10.0
0
No
Yes
102
End Loop
274
0.0
0
No
Yes
103
Go Sub
287
0.0
10
No
Yes
104
Endif
281
0.0
0
No
Yes
105
Select Act Cols
283
0.0
0
No
Yes
106
Go Sub C
294
0.0
2
No
Yes
107
Go Sub
287
0.0
11
No
Yes
108
Time Stamp
396
0.0
31
No
Yes
109
Depro Htr Wait
169
0.0
0
No
Yes
110
Time Stamp
396
0.0
32
No
Yes
111
Wait Coil Temp
261
0.0
0
No
Yes
112
Set Coil Temp
260
0.0
0
No
Yes
113
Time Stamp
396
0.0
33
No
Yes
114
Xfer Coil C
168
12.0
1
Yes
No
115
Xfer Coil C
168
20.0
0
No
No
116
Go Sub B
293
0.0
2
No
Yes
117
Xfer Coil B
167
12.0
1
Yes
No
118
Xfer Coil B
167
20.0
0
No
No
119
Go Sub A
292
0.0
2
No
Yes
D-50 ABI 3948 System Function List and Other Cycles
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
1
Yes
No
120
Xfer Coil A
166
12.0
121
Xfer Coil A
166
20.0
0
No
No
122
Time Stamp
396
0.0
34
No
Yes
123
Clv Owns Depro
286
0.0
0
No
Yes
124
Go Sub
287
0.0
10
No
Yes
125
Select Pur Cols
282
0.0
0
No
Yes
126
If Any Act Cols
291
0.0
0
No
Yes
127
Set Pres Reg 3
303
0.0
9
No
Yes
128
Gas to Pur Cols
203
60.0
0
No
Yes
129
Set Pres Reg 3
303
0.0
5
No
Yes
130
PurLowBlk Flush
218
2.0
0
No
Yes
131
Begin Loop
273
0.0
2
No
Yes
132
AcAcid to Waste
202
2.0
0
No
Yes
133
AcAcid to Purs
198
3.0
0
No
Yes
134
AcAcid to Purs
198
30.0
0
Yes
Yes
135
Gas to Pur Cols
203
25.0
0
No
Yes
136
ACN to PurWaste
177
2.0
0
No
Yes
137
PurLowBlk Flush
218
5.0
0
No
Yes
138
End Loop
274
0.0
0
No
Yes
139
Gas to Pur Cols
203
15.0
0
No
Yes
140
Go Sub A
292
0.0
7
No
Yes
141
Go Sub B
293
0.0
7
No
Yes
142
Go Sub C
294
0.0
7
No
Yes
143
Go Sub
287
0.0
4
No
No
144
Go Sub
287
0.0
3
No
Yes
145
Go Sub
287
0.0
4
No
Yes
146
Go Sub
287
0.0
3
No
Yes
147
Begin Loop
273
0.0
5
No
Yes
148
Go Sub
287
0.0
9
No
Yes
149
End Loop
274
0.0
0
No
Yes
150
Begin Loop
273
0.0
3
No
No
151
Go Sub
287
0.0
4
No
No
152
Go Sub
287
0.0
3
No
Yes
153
End Loop
274
0.0
0
No
No
154
20%ACN to Waste
197
4.0
0
No
Yes
155
20%ACN to Purs
193
6.0
0
No
Yes
156
20%ACN to Purs
193
15.0
0
Yes
Yes
157
Go Sub
287
0.0
10
No
Yes
158
Set Pres Reg 3
303
0.0
4
No
Yes
159
PurLowBlk Flush
218
2.0
0
No
Yes
160
Gas to Pur Cols
203
10.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-51
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
281
0.0
0
No
Yes
161
Endif
162
Time Stamp
396
0.0
35
No
Yes
163
Select Act Cols
283
0.0
0
No
Yes
164
Go Sub A
292
0.0
1
No
Yes
165
Go Sub A
292
0.0
12
No
Yes
166
SC Close
246
0.0
0
No
Yes
167
SC Needle Down
248
0.0
0
No
Yes
168
Gas to PurCol A
204
7.0
0
No
Yes
169
Pur A to UV
211
10.0
3
Yes
Yes
170
Pur A to UV
211
20.0
0
No
Yes
171
Go Sub A
292
0.0
14
No
Yes
172
Go Sub A
292
0.0
15
No
Yes
173
Go Sub B
293
0.0
1
No
Yes
174
Go Sub B
293
0.0
12
No
Yes
175
SC Next
244
0.0
0
No
Yes
176
Gas to PurCol B
205
7.0
0
No
Yes
177
Pur B to UV
212
10.0
3
Yes
Yes
178
Pur B to UV
212
20.0
0
No
Yes
179
Go Sub B
293
0.0
14
No
Yes
180
Go Sub B
293
0.0
16
No
Yes
181
Go Sub C
294
0.0
1
No
Yes
182
Go Sub C
294
0.0
12
No
Yes
183
SC Next
244
0.0
0
No
Yes
184
Gas to PurCol C
206
7.0
0
No
Yes
185
Pur C to UV
213
10.0
3
Yes
Yes
186
Pur C to UV
213
20.0
0
No
Yes
187
Go Sub C
294
0.0
14
No
Yes
188
Go Sub C
294
0.0
17
No
Yes
189
Go Sub
287
0.0
12
No
Yes
190
SC Next
244
0.0
0
No
Yes
191
SC Open
245
0.0
0
No
Yes
192
Time Stamp
396
0.0
36
No
Yes
193
Set Pres Reg 3
303
0.0
5
No
Yes
194
Gas to Pur Cols
203
45.0
0
Yes
Yes
195
Gas to Pur Cols
203
10.0
0
No
Yes
196
Go Sub
287
0.0
3
No
Yes
197
Go Sub
287
0.0
8
No
Yes
198
Go Sub
287
0.0
3
No
Yes
199
Time Stamp
396
0.0
37
No
Yes
200
Goto End
290
0.0
0
No
Yes
201
Sub Label
289
0.0
1
No
Yes
D-52 ABI 3948 System Function List and Other Cycles
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
303
0.0
6
No
Yes
202
Set Pres Reg 3
203
SC Waste
242
0.0
0
No
Yes
204
H2O to PurWaste
187
5.0
0
No
Yes
205
H2O to UV
214
5.0
0
No
Yes
206
SC Block Flush
222
5.0
0
No
Yes
207
Wait
259
10.0
0
No
Yes
208
UV Reading
216
2.0
0
No
Yes
209
Flsh UV toWaste
217
6.0
0
No
Yes
210
H2O to UV
214
1.0
0
No
Yes
211
SC Block Flush
222
10.0
0
No
Yes
212
Xfer UV to SC
215
10.0
0
No
Yes
213
Return Sub
288
0.0
0
No
Yes
214
Sub Label
289
0.0
2
No
Yes
215
Set Pres Reg 1
301
0.0
12
No
Yes
216
Set Pres Reg 3
303
0.0
12
No
Yes
217
ACN to PurWaste
177
0.5
0
No
Yes
218
PurLowBlk Flush
218
3.0
0
No
Yes
219
Pur Xfer Rinse
171
2.0
0
No
Yes
220
Pur Xfer Flush
172
15.0
0
No
Yes
221
Set Pres Reg 1
301
0.0
8
No
Yes
222
DepLowBlk Flush
153
2.0
0
No
No
223
Return Sub
288
0.0
0
No
No
224
Sub Label
289
0.0
3
No
Yes
225
Set Pres Reg 3
303
0.0
12
No
Yes
226
Back Flush Purs
207
6.0
0
No
Yes
227
Back Flush Purs
207
45.0
0
Yes
Yes
228
Back Flsh Pur A
208
6.0
0
No
Yes
229
Back Flsh Pur B
209
6.0
0
No
Yes
230
Back Flsh Pur C
210
6.0
0
No
Yes
231
Begin Loop
273
0.0
2
No
Yes
232
Gas to Pur Cols
203
1.0
0
No
Yes
233
Back Flush Purs
207
4.0
0
No
Yes
234
End Loop
274
0.0
0
No
Yes
235
Return Sub
288
0.0
0
No
Yes
236
Sub Label
289
0.0
4
No
Yes
237
Set Pres Reg 3
303
0.0
9
No
Yes
238
H2O to Pur Cols
183
3.0
0
No
Yes
239
H2O to Pur Cols
183
15.0
0
Yes
Yes
240
Gas to Pur Cols
203
15.0
0
Yes
Yes
241
Gas to Pur Cols
203
6.0
0
No
Yes
242
Return Sub
288
0.0
0
No
Yes
ABI 3948 System Function List and Other Cycles D-53
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
289
0.0
5
No
Yes
243
Sub Label
244
Set Pres Reg 3
303
0.0
4
No
Yes
245
ACN to Pur Cols
173
4.0
0
No
Yes
246
ACN to Pur Cols
173
15.0
0
Yes
Yes
247
Gas to Pur Cols
203
15.0
0
Yes
Yes
248
Gas to Pur Cols
203
6.0
0
No
Yes
249
Return Sub
288
0.0
0
No
Yes
250
Sub Label
289
0.0
6
No
Yes
251
Crude A to UV
135
12.0
1
Yes
Yes
252
Crude A to UV
135
20.0
0
No
Yes
253
Crude B to UV
136
12.0
1
Yes
Yes
254
Crude B to UV
136
20.0
0
No
Yes
255
Crude C to UV
137
12.0
1
Yes
Yes
256
Crude C to UV
137
20.0
0
No
Yes
257
Return Sub
288
0.0
0
No
Yes
258
Sub Label
289
0.0
7
No
Yes
259
Set Pres Reg 3
303
0.0
12
No
Yes
260
ACN to PurWaste
177
2.0
0
No
Yes
261
PurLowBlk Flush
218
5.0
0
No
Yes
262
Set Pres Reg 3
303
0.0
6
No
Yes
263
Back Flush Purs
207
15.0
0
No
Yes
264
Back Flush Purs
207
45.0
3
Yes
Yes
265
Set Pres Reg 3
303
0.0
12
No
Yes
266
Back Flush Purs
207
15.0
0
No
Yes
267
Return Sub
288
0.0
0
No
Yes
268
Sub Label
289
0.0
8
No
Yes
269
Set Pres Reg 3
303
0.0
6
No
Yes
270
ACN to Pur Cols
173
4.0
0
No
Yes
271
ACN to Pur Cols
173
15.0
0
Yes
Yes
272
Gas to Pur Cols
203
15.0
0
Yes
Yes
273
Gas to Pur Cols
203
3.0
0
No
Yes
274
Set Pres Reg 3
303
0.0
8
No
Yes
275
Begin Loop
273
0.0
2
No
Yes
276
H2O to Pur Cols
183
4.0
0
No
Yes
277
H2O to Pur Cols
183
15.0
0
Yes
Yes
278
Gas to Pur Cols
203
15.0
0
Yes
Yes
279
Gas to Pur Cols
203
3.0
0
No
Yes
280
End Loop
274
0.0
0
No
Yes
281
Set Pres Reg 3
303
0.0
12
No
Yes
282
Gas to Pur Cols
203
8.0
0
No
Yes
283
Return Sub
288
0.0
0
No
Yes
D-54 ABI 3948 System Function List and Other Cycles
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
289
0.0
9
No
Yes
284
Sub Label
285
Set Pres Reg 3
303
0.0
4
No
Yes
286
PurLowBlk Flush
218
2.0
0
No
Yes
287
TFA to Pur Cols
188
6.0
0
No
Yes
288
TFA to Pur Cols
188
25.0
0
Yes
Yes
289
Wait
259
15.0
0
No
Yes
290
Begin Loop
273
0.0
2
No
Yes
291
TFA to PurCol A
189
1.0
999
No
Yes
292
TFA to PurCol B
190
1.0
999
No
Yes
293
TFA to PurCol C
191
1.0
999
No
Yes
294
Wait
259
15.0
0
No
Yes
295
End Loop
274
0.0
0
No
Yes
296
Begin Loop
273
0.0
6
No
Yes
297
Gas to PurCol A
204
0.6
0
No
Yes
298
Gas to PurCol B
205
0.6
0
No
Yes
299
Gas to PurCol C
206
0.6
0
No
Yes
300
Pur Vent
220
2.0
0
No
Yes
301
Wait
259
23.0
0
No
Yes
302
End Loop
274
0.0
0
No
Yes
303
Set Pres Reg 3
303
0.0
10
No
Yes
304
Tggle Vlves On
262
0.0
70
No
Yes
305
Back Flsh Pur A
208
12.0
0
No
Yes
306
Back Flsh Pur B
209
12.0
0
No
Yes
307
Back Flsh Pur C
210
12.0
0
No
Yes
308
Set Pres Reg 3
303
0.0
12
No
Yes
309
Back Flsh Pur A
208
10.0
0
No
Yes
310
Back Flsh Pur B
209
10.0
0
No
Yes
311
Back Flsh Pur C
210
10.0
0
No
Yes
312
Gas to Pur Cols
203
2.0
0
No
Yes
313
Back Flush Purs
207
8.0
0
No
Yes
314
Tggle Vlves Off
263
0.0
70
No
Yes
315
Return Sub
288
0.0
0
No
Yes
316
Sub Label
289
0.0
10
No
Yes
317
H2O to PurWaste
187
1.0
0
No
Yes
318
Set Pres Reg 3
303
0.0
12
No
Yes
319
PurLowBlk Flush
218
6.0
0
No
Yes
320
Return Sub
288
0.0
0
No
Yes
321
Sub Label
289
0.0
11
No
Yes
322
Set Rmp Fxn Sns
265
0.0
10
No
Yes
323
Set Rmp Fxn Trp
266
0.0
5
No
Yes
324
Set Rmp Fxn Chk
267
0.0
5
No
Yes
ABI 3948 System Function List and Other Cycles D-55
Table D-3 Pur v4.40 Biotin Cycle
STEP
FUNCTION NAME
NUM
TIME
MISC
SNS
SAFE
325
Set Rmp Fxn Dly
268
0.0
60
No
Yes
326
Return Sub
288
0.0
0
No
Yes
327
Sub Label
289
0.0
12
No
Yes
328
Set Pres Reg 3
303
0.0
10
No
Yes
329
SC Waste
242
0.0
0
No
Yes
330
H2O to PurWaste
187
3.0
0
No
Yes
331
H2O to UV
214
3.0
0
No
Yes
332
PurLowBlk Flush
218
5.0
0
No
Yes
333
SC Block Flush
222
5.0
0
No
Yes
334
Flsh UV toWaste
217
8.0
0
No
Yes
335
Set Pres Reg 3
303
0.0
5
No
Yes
336
Flsh UV toWaste
217
3.0
0
No
Yes
337
Return Sub
288
0.0
0
No
Yes
338
Sub Label
289
0.0
13
No
Yes
339
Return Sub
288
0.0
0
No
Yes
340
Sub Label
289
0.0
14
No
Yes
341
ACN to Pur Cols
173
4.0
0
No
Yes
342
ACN to Pur Cols
173
15.0
0
Yes
Yes
343
H2O to Pur Cols
183
10.0
0
No
Yes
344
Set Pres Reg 3
303
0.0
12
No
Yes
345
PurLowBlk Flush
218
5.0
0
No
Yes
346
Set Pres Reg 3
303
0.0
6
No
Yes
347
PurLowBlk Flush
218
2.0
0
No
Yes
348
Return Sub
288
0.0
0
No
Yes
349
Sub Label
289
0.0
15
No
Yes
350
UV Reading
216
2.0
1
No
Yes
351
Xfer UV to SC
215
15.0
0
No
Yes
352
Return Sub
288
0.0
0
No
Yes
353
Sub Label
289
0.0
16
No
Yes
354
UV Reading
216
2.0
2
No
Yes
355
Xfer UV to SC
215
15.0
0
No
Yes
356
Return Sub
288
0.0
0
No
Yes
357
Sub Label
289
0.0
17
No
Yes
358
UV Reading
216
2.0
3
No
Yes
359
Xfer UV to SC
215
15.0
0
No
Yes
360
Return Sub
288
0.0
0
No
Yes
361
End of Cycle
272
0.0
0
No
Yes
D-56 ABI 3948 System Function List and Other Cycles
Instrument Plumbing
Diagram
E
E
Instrument Plumbing Diagram E-1
Limited Warranty
Statement
F
F
Applied Biosystems warrants to the customer that, for a period ending on the earlier of
two (2) years from the completion of installation or twenty-seven (27) months from the
date of shipment to the customer (the “Warranty Period”), the ABI 3948 Nucleic Acid
Synthesis and Purification System purchased by the customer (the “Instrument”) will
be free from defects in material and workmanship, and will perform in accordance with
the specifications set forth in the ABI 3948 System Data Sheet (the “Specifications”).
During the Warranty Period, if the Instrument's hardware becomes damaged or
contaminated or if the Instrument otherwise fails to meet the Specifications, Applied
Biosystems will repair or replace the Instrument so that it meets the Specifications, at
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Limited Warranty Statement F-1
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F-2 Limited Warranty Statement
Index
Numerics
3' phosphorus 3-3
4X12 configuration rack
standard white 2-22
A
ABI 3948
calculation of
concentration 3-25
computer controlled 2-6
Abort
defined 1-4
Abort command
definition 4-21
procedure 4-22
absorbance
reported in ODUs (Optical Density
Units) and picomoles 2-5
acetonitrile
wash before detritylation 3-9
Alternative Chemistries 3-28
aminomethyl linker
attached to support material 3-4
ammonium hydroxide, use in
cleavage 3-16
application memory setting
default 4-10
Applications
used to create Multi-Order
files 4-41
Apply Button 6-14
Automation
Chemistry Processes 2-4
Auto-res OK 0.01 PSI
parameter 4-28
Auto-Resume feature
Instrument Preferences command
auto-resume feature 4-27
Auto-resume Minutes
parameter 4-28
B
B+ Tet Calibration
contents of view 6-8
base deprotection
guidelines 3-17
Baseline absorbance
setting by the system
Beer’s Law 3-22
Begin and End Procedures
defined 1-4
3-24
benzoyl
protection group 3-7
Block functions
gas flush, pressurization, and
rinse A-22
priming delivery lines A-21
priming for Cleavage
Columns A-21
priming for Purification
Columns A-22
Bottle change functions A-20
bottle procedure
editing 6-22
C
Calculation
of concentration (pmol/µL) 3-25
Capping
characteristics 3-13
process 3-13
capping 3-13–??, 3-13–3-14
by acetylation 3-13
highly recommended 3-13
needed because coupling is not
always quantitative 3-13
reactive chains not affected by
capping 3-13
Change Name command
definition 4-21
procedure 4-26
Change Passwords command
definition 4-21
Check Illegal Values parameter 4-29
chemical delivery system
pressure driven 2-3
Chemistries
alternative 3-28
Chemistry
of choice 3-7
progress during a run 5-12
solid phase synthesis 3-3
chemistry
progress of 5-12
Chemistry Panes
description of 5-12
Chemistry Processes
automation 2-4
First Four 2-4
Chemistry protocols
description C-2
Chemistry reactions
details of four reactions 3-6
Chemistry Stages A-3
Cleavage
module 2-4
process of 2-15
cleavage and
deprotection 3-16–3-17
removal of protecting
groups 3-17
Cleavage Cycle parameter 5-9
Cleavage functions
delivery to cleavage
columns A-17
gas flushes and
pressurization A-17
oligonucleotide transfer A-18
water delivery to deprotection
coil A-18
cleavage jaw sensors 5-16
Cleavage module
components 2-7
location and description 2-15
Cleavage reaction 3-16
Close All Synth Orders
command 4-3
definition 4-3
Close command 4-3
Coil Cool Secs (261) parameter 4-28
collection needle
waste bottles 2-22
Column turntable
purpose of 2-7
Command-Key commands 4-4
Command-key equivalents (hot keys)
Edit menu 4-17
File menu 4-4
Commands and capabilities
overview 4-1
Communication View
initial database window 5-6
concentrated aqueous ammonium
hydroxide
used to cleave the
oligonucleotide 2-4
controller
also fills out Synthesis
Orders 2-3
real time control 2-3
Copy from Button 6-14
Coupling
characteristics 3-12
Index-1
coupling 3-11–3-12, ??–3-12
effect of excess of
phosphoramidite 3-12
mixed sequence probes 3-12
Nucleophilic attack by
tetrazole 3-12
reactivity order for cyanoethyl
phosphoramidites 3-12
Creating
Synthesis Orders 4-38
Creating a User Function
procedure 6-5
Criteria
for converting ODU to mass or
concentration 3-22
Critical messages C-42
crude oligonucleotides
with the 5´ trityl group either on or
off 2-4
Current date and time
where displayed 5-15
cuvette 2-20
cyanoethyl
protecting group 3-8
Cyanoethyl phosphoramidite
four functional groups 3-8
cyanoethyl phosphoramidites
reactivity order 3-12
cycle
definition of A-3
Cycle and Procedure Pop-Up Menus
Edit views 6-13
D
denaturing media 3-20
Deprotect Mins (169)
parameter 4-28
Deprotect Temp (170)
parameter 4-28
Deprotection
module 2-4
process of 2-17
time and temperature 2-4
deprotection
base 3-17
phosphate 3-16
deprotection coil sensors 5-17
Deprotection module
components 2-7, 2-16
location 2-16
Depurination
purines susceptable to 3-9
depurination
minimization of 3-10
Detritylation
chemistry process 3-9
diisopropylamino functional
group 3-8
dimethoxytrityl group
removal 3-5
Index-2
DNA products
description of elution 2-4
DNA purification 3-20
DNA storage
period of stability 3-27
DNA Synthesis Cycle
four steps 3-3
Export Sequence command
definition 4-3
Ext. Coefficient 5, 6, 7, and 8
description 4-28
Extinction coefficient
major contributors to 3-22
Extinction coefficients
for fluorescent dye labels 3-26
E
F
Edit Begin Procedure View 6-20
Edit Bottle Procedure View 6-22
failure sequences 3-13
purpose of view 6-22
File commands
Import/Export 4-15
Edit Cleavage Cycle View
purpose of view 6-18
Open 4-8
Edit commands
Save a Copy In 4-13
Find Same 4-18
Send Copy to Synthesizer 4-14
Read Selection 4-17
Send to Synthesizer 4-13
Replace command 4-18
File menu 4-3–??
list of commands 4-3
Edit Cycle and Procedure interface
Buttons 6-14
list of tasks 4-3
entry fields 6-16
final detritylation
a trityl flush 3-9
interface elements 6-12
Edit End Procedure View
Find command 4-18
editing 6-21
Find Same command
procedure 4-18
Edit Function View
scrollable lists 6-4
Find/Find Same commands
definition 4-17
Edit menu ??–4-19
list of commands 4-17
Flow path
ACN to Column A
Edit Purification Cycle View
(illustration) A-9
purpose of view 6-19
edit sequence 4-18
flowcell
1 cm pathlength quartz 3-23
Edit Synthesis Cycle View
purpose of 6-17
Function names A-7
Edit Views, all
functional groups
same general layout 6-12
on phosphoramidites 3-8
Electromechanical and electrical
Functions
drivers 2-6
three fundamental types A-7
embedded software 2-6
Time and Misc field entries A-7
End Row on Jaw Leak
tracing flow paths A-8
parameter 4-29
functions
printing 6-5
End sequence
with A, B, C or T on the 3´
Functions in cycles
end 4-7
description C-2
entering sequences
as mixed bases 4-39
G
Entry fields
Gas pressure sensors/sensor
Edit Cycle and Procedure
drivers 2-6
interface 6-16
General activity messages C-40
Execute Button 6-14
Generate Run File command 4-16
Exercising Manual Control
definition 4-3
of a Function
Generating Synthesis Orders
Manual Control View
from Multi-Order files 4-42
exercising control of a
function 6-4
H
exocyclic amines 3-5
Hardware
Export command 4-15
types of 2-7
export files
Hierarchical messages C-43
names and icons 4-15
high throughput 2-4
Export Procedure icons 4-15
I
Import command 4-15
Import Sequence command
definition 4-3
Import/Export
discussion 4-15
Importing and Exporting Sequences
Synthesis Orders 4-39
Importing Synthesis Orders
process 5-21
initial Chemistry Pane view 5-12
Insert Button 6-15
instrument control
real time 2-3
Instrument Dip Switches
Instrument Preferences
Instrument Dip Switches
parameters 4-29
Instrument Messages C-40
Instrument plumbing diagram E-1
Instrument Preferences command
definition 4-21
purpose of 4-27
Setup Variables 4-28
Instrument Test View
activating tests 6-8
tests performed 6-7
Interrupt
defined 1-4
Interrupt command 4-22
definition 4-21
iodine reagent
in oxidation 3-15
isobutyryl
protection group 3-7
J
Jaw Leak Test
in PSI parameter 4-28
Jaw mechanism
illustration and detailed
description 2-11
purpose of 2-7
K
key terms
table of
1-4
L
Label View
information in individual
labels 4-36
Label View information
in the five columns 4-36
Leak OK 0.01 PSI
parameter 4-28
Limited warranty statement F-1
Liquid sensors 2-6
conventions 5-16
list of open windows 4-30
Log Dry Sensor Fxns
parameter 4-29
Low pressure mercury lamp
low-pressure mercury lamp
(P/N) 2-20
3-23
M
Monomers
other than standard
phosphoramidite 3-28
Multi-Order files
types of applications used to
create 4-41
N
Man Cont Jaw Testing
parameter 4-29
Manual Control
Creating a User Function 6-5
exercises valve functions A-10
printing functions 6-5
Manual Control View
viewing a function valve list 6-4
manual volumes, two
purposes of 1-1
Maxam-Gilbert sequencing
reference to 3-9
Method of synthesis
Phosphoramidite 2-3
Microphone-only messages C-42
Misc (Miscellaneous) Procedures View
purpose of view 6-23
Miscellaneous hardware
functions A-24
Miscellaneous Procedures C-37
mixed base entry 4-39
Mixed sequence probes
synthesis of 3-12
Mnfg. Jaw Test Mode
parameter 4-29
modify
sequence 4-18
module 2-4
Molar extinction coefficent 3-22
Monitor Chemistry View
interface information 5-11
sequence pane
description 5-13
Status and System
Message 5-13
three chemistry panes 5-12
Monitor Chemistry view
description of three chemistry
panes 5-12
general description 5-11
using information 5-14
Monitor Instrument View
other
parameters/conditions 5-
18
Monitor Run View
general information 5-19
types of information
provided 5-19
monitoring
valves 5-15
monitoring chemistry 5-14
name
changing the synthesizer 4-26
New Synthesizer Order command
definition 4-3
NMI reagent
in capping 3-13
No Flow To Open Jaws
parameter 4-29
non-valve hardware functions
pressure regulating A-23
nucleosides
phosphoramidite 3-7
Nucleotide 3´-hydroxyl
attachment to support 3-4
O
ODU
as a measure of
concentration 3-22
criteria for converting to mass or
concentration 3-22
definition 3-22
ODU as the Unit of Measure
definitions which apply 3-22
ODU Measurement
optimum measurement
criteria 3-22
oligonucleotide production
description of process 2-3
oligonucleotide quantitation 3-22
oligonucleotide yield
automatic measurement 3-23
oligonucleotides
methods of purification 3-20
storage for later use 3-27
types to avoid storing 3-27
OneStep column
contains the 3´-terminal
nucleoside 2-4
illustration and detailed
description 2-9
moves between three
modules 2-4
three of four chemistry processes
occur within 2-4
OneStep column turntable
illustration and detailed
description 2-10
OneStep™ Column
four types 2-7
OneStep™ column 2-3
Index-3
Open command 4-8
definition 4-3
Open Synthesizer command 4-3
definition 4-3
Opening an Oligo Labels window for
printing
alternate way 4-35
oxidation 3-15
Oxidation after capping
importance of 3-15
Oxidation reaction 3-15
P
PAGE and HPLC
use for purification 3-20
Page Setup command
definition 4-3
Parallel and serial User Functions
use together A-13
Pause After
defined 1-4
Pause After Command
programming a pause 4-23
Pause After command
definition 4-21
Pause On Jaw Leak
parameter 4-29
Pause On Sensor Fail
parameter 4-29
Phosphate deprotection 3-16
phosphoramidite method of
oligonucleotide synthesis
characteristics 3-7
Phosphoramidite method of synthesis
overview 3-6
phosphoramidite method of
synthesis 2-3
phosphoramidite nucleosides
Figure 3-7
Phosphoramidite nucleotides
functional groups 3-8
structures and molecular
weights 3-7
phosphoramidite sensors 5-17
phosphoramidites
function groups 3-8
phosphoramidites and tetrazole
delivered simultaneouslyl during
coupling 3-11
polystyrene
used as solid support for DNA
synthesis 3-4
Polystyrene support
highly cross-linked 3-3
Power Fail View
purpose of 6-6
Pressure regulating functions A-23
Pressure Regulation and Control
(PRC) module
characteristics 2-12
Index-4
Pressure Regulation and Control
module (PRC)
purpose of 2-7
Printing labels from RunFiles
alternate way 4-35
information in individual
labels 4-36
procedure
definition of A-3
types of A-3
Procedures
bottle change (Manual
Dilution) C-36
End C-34
miscellaneous C-37
phosphoramidite
autodilution C-36
Start Up C-32
procedures
for beginning synthesis C-32
protecting groups
removal 3-17
protection groups 3-7
Protocol
defined 1-4
Protocol Name parameter 5-9
Purification 2-4
characteristics of 3-20
other methods 3-20
process of 2-19
Purification and quantitation functions
collecting and quantitating
samples A-19
delivery of reagents to purification
columns/UV
detector A-18
rinsing and flushing deprotection
columns A-19
Purification biotin cycle
listing D-48
purification coil sensors 5-17
Purification Cycle parameter 5-10
Purification dye cycle
listing D-39
Purification module
components 2-7
location and description 2-18
Purification stages 3-20
purification step
omitting 3-5
purified oligonucleotides
must be synthesized with the 5´
trityl group on 2-4
Q
Quantitation
general description 2-5
Quantitation module 2-20
components 2-7
quantitation of oligonucleotides
by UV spectroscopy 3-22
R
Racks
two types of 2-22
racks
outside dimensions 2-22
reaction column chamber
OneStep column 2-3
reactive chains
still trityl blocked 3-13
Read Selection command 4-17,
4-40
definition 4-17
procedure 4-17
red rack
optional 2-22
Reference manual
listing of chapter contents 1-2
Refrigerated storage
in solution 3-27
Regular reports messages C-40
Replace 4-18
Replace command
procedure 4-18
Replace/Replace Same commands
definition 4-17
Reserved parameter 4-29
Resume command 4-22
definition 4-21
Run Protocol View 5-10
Cleavage Cycle parameter 5-9
Protocol Name parameter 5-9
Synthesis Cycle parameter 5-9
used to create protocols 5-9
Run Setup View
assigning chemistry 5-25
autoSorting 5-25
description of Open Dialog box
buttons 5-22
information provided for a selected
sequence 5-24
list of Menus and Buttons 5-8
list of tasks performed
accessing 5-7
selecting orders for the next
run 5-23
RunFiles
list of types of information 4-32
S
Safe/Sensor Buttons 6-15
Sample collection
general description 2-5
unattended collection of 48 DNA
samples 2-5
Sample Collection module
components 2-7
components and
illustration 2-21
needle assembly 2-21
Sample Labeling feature
procedure 4-33
Sample racks
characteristics 2-22
sample vials
sample volume 2-5
Save & Reopen command 4-3
definition 4-3
Save a Copy In Command
discussion 4-13
Save a Copy In command 4-13
definition 4-3
Save and Reopen command
discussion 4-14
Save As command 4-3
definition 4-3
Save command 4-3
definition 4-3
Scaling factor 3-24
Select All command
definition 4-17
Send Copy to Synthesizer
command 4-14
definition 4-3
Send to Synthesizer command 4-3
definition 4-3
types of files sent 4-13
Sensor User Function names
components of A-12
Sensor-Controlled User Function
example A-14
Sensors
general characteristic A-11
location of A-11
names and locations A-13
retries A-12
use of parallel with serial Sensor
User Functions A-13
septum sheet
purpose of 2-5
where located 2-5
sequence
import
export 4-15
Sequence entry
Synthesis Orders 4-6
sequence modification 4-18
sequence pane description
Monitor Chemistry View 5-13
Setup Choices
Instrument Preferences
Setup Choices 4-29
Setup Variables 4-28
Show Clipboard command
definition 4-17
Solid support
characteristics 3-3
Solid–phase synthesis chemistry 3-3
Special functions
overview of B-2
Standard Cleavage/Deprotection Cycle
listing C-12
summary C-9
Standard Purification Cycle
listing C-23
summary C-18
Standard Synthesis Cycle
summary C-3
Standard Synthesis Cycle listing C-5
Status and System Messages
Monitor Chemistry View 5-13
Storage
frozen in 20% acetonitrile 3-27
guidelines 3-27
of crudes 3-27
other liquid media 3-27
string substitutions 4-18
succinate ester bond 3-4
Support
characteristics 3-4
support material
highly cross-linked
polystyrene 3-4
Synchronize Clocks command 4-24
definition 4-21
Synthesis
module 2-4
process 3-3
process of 2-14
synthesis
completion of 3-5
Synthesis column functions
flushing and back flushing A-16
Synthesis Cycle
concluding steps 3-5
Synthesis Cycle parameter 5-9
synthesis jaw sensors 5-16
synthesis manifold sensors 5-17
Synthesis Module
components 2-7
Synthesis module
location and description 2-13
Synthesis Order
sequence entry 4-39
Synthesis Order command
description of fields 4-5
types of information 4-5
Synthesis Order information
provided for a selected
sequence 5-24
Synthesis Orders
actions after Sequence
Entry 4-40
importing and exporting
sequences 4-39
information contained
within 4-38
ways of creating 4-38
Synthesis scale
description C-2
Synthesis valve functions A-15
Synthesis Window
editing 5-1
Synthesizer Commands
list of definitions 4-21
Synthesizer commands
Synchronize Clocks command
procedure 4-24
Synthesizer menu 4-21–??
Abort 4-22
Change Name 4-26
Interrupt 4-22
Resume 4-22
Synchronize clocks 4-24
Synthesizer Window
accessing by password 5-6
general information 5-4
keyboard shortcuts 5-5
list of views 5-4
Synthesizer window
list of views 4-11
Synthesizer Window views
list of 4-11
System Functions A-7
System messages C-38
categories of messages C-39
critical messages C-42
hierarchical messages C-43
message conventions C-39
Microphone-only
messages C-42
regular reports messages C-40
types of messages C-38
T
TCA
in detritylation 3-9
three stations
concurrent use of A-4
trichloroacetic acid 3-9
for DMT removal 3-5
trityl
protecting group 3-8
trityl group
as handle for purification 3-5
Turntable control functions A-23
turntable positions (A, B, C) 5-16
types of runs
pre-programmed or custom 2-3
Types of storage 2-6
Index-5
U
Use Sounds command
definition 4-17
User 4x12 Tube Rack
parameter 4-29
User bulletins
contents and purpose 1-4
User cycles
description C-2
User Functions A-7
general description A-12
User functions A-20
UV detection system
description 2-20
UV detector sensor 5-17
UV flowcell
illustration 3-23
UV hardware 3-23
UV scaling factor 3-24
UV spectroscopy 3-22
UV-light source
254 nm 2-20
V
Valve Functions
creating your own A-10
types of A-10
uses of A-10
Valve functions
list of function categories A-15
valve status
off or on 5-15
Valves
list of valves A-5
vial septum
chemically inert 2-5
Viewing a function valve list 6-4
W
waste bottles
location of bottles for collection
needle rinse 2-22
wetted components
chemically inert 2-11
Window menu 4-30
description 4-30
X
Xfer From Coil (261) parameter
Xfer Into Coil (260) parameter
Index-6
4-28
4-28
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