Download ABI 391 manual

PCR-MATE EP MODEL 391
DNA SYNTHESIZER
User’s Manual
© Copyright 2002, Applied Biosystems. All rights reserved.
PCR-MATE, OPC, Aminolink 2, and Syncom are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. The
Polymerase Chain Reaction (PCR) process is covered by Cetus Corporation’s U.S. Patent Number 4,683,202. No license to use the PCR process is given
or implied by Applied Biosystems Cetus’ licensing of PCR-MATE.
Teflon is a trademark of the E.I. DuPont de Neumors Company.
Parafilm is a trademark of the American Can Company.
Drierite is a trademark of the W.A. Hammond Drierite Company.
Swagelock is a trademark of the Swagelock Company.
HOW TO START
Begin by reading Section 1, Introduction. It contains vital information and will help you understand how the manual is
organized and how to use it. Next, refer to Section 2, Operation, for instructions on how to prepare and start a synthesis.
Contents
Section 1:
Introduction
How To Use This Manual .....................................................................................................1-2
General Introduction .............................................................................................................1-3
How to Get Help ...................................................................................................................1-5
Contacting Technical Support .........................................................................................1-5
To Contact Technical Support by E-Mail.........................................................................1-5
Hours for Telephone Technical Support..........................................................................1-5
To Contact Technical Support by Telephone or Fax.......................................................1-6
To Reach Technical Support Through the Internet .........................................................1-8
To Obtain Documents on Demand..................................................................................1-9
User Attentions ...................................................................................................................1-10
Material Safety Data Sheets (MSDS) .................................................................................1-10
Text Conventions ...............................................................................................................1-11
Section 2:
Operation
Power On .............................................................................................................................2-2
Pre-synthesis Check List ......................................................................................................2-3
How to Prepare Phosphoramidites.......................................................................................2-4
How to Dissolve Phosphoramidites.................................................................................2-5
How to Store Dissolved Phosphoramidites .....................................................................2-6
How to Install Reagent Bottles .............................................................................................2-7
How to Install the Phosphoramidites (bottles 1-5)...........................................................2-7
How to Install Bottles 9-15...............................................................................................2-8
How to Install Bottle 18....................................................................................................2-8
How to Begin a Synthesis ....................................................................................................2-9
How to Install the Column .............................................................................................2-12
Post Synthesis....................................................................................................................2-13
Manual Deprotection and Cleavage ...................................................................................2-13
Introduction....................................................................................................................2-13
Materials and Methods ..................................................................................................2-14
Methods.........................................................................................................................2-16
How to Abort a Synthesis ...................................................................................................2-18
About the Synthesis Reagents and Solvents .....................................................................2-19
i
Applied Biosystems
How to Store Reagents .................................................................................................2-19
Manual Deprotection and Cleavage Reagents..............................................................2-22
About Deoxyinosine ......................................................................................................2-23
How to Change an Argon Tank ..........................................................................................2-25
How to Change the Waste Bottle .......................................................................................2-25
How to Perform the Flow Test Procedure ..........................................................................2-26
Aminolink 2™ Reagent.......................................................................................................2-30
Introduction....................................................................................................................2-30
Chemical Description ....................................................................................................2-30
How to Use Aminolink 2 .....................................................................................................2-32
Analysis and Purification of Aminolink™-Oligonucleotides ................................................2-32
3'-End-Labeling .............................................................................................................2-32
Applications ........................................................................................................................2-34
General Protocol for Tag/Labeling Substrate ................................................................2-34
How to Use OPC (Oligonucleotide Purification Cartridge) .................................................2-35
Introduction....................................................................................................................2-35
Protocol for Lysine Pre-treatment of Longmers (>70 bases) ........................................2-37
OPC Purification Protocol...................................................................................................2-38
Solutions Needed ..........................................................................................................2-38
References .........................................................................................................................2-40
Section 3:
Software Menu Descriptions
Introduction...........................................................................................................................3-2
The MAIN and MENU Soft Keys .....................................................................................3-3
Software Abbreviations and Symbols...................................................................................3-4
The Main Menu ....................................................................................................................3-5
Summary of Main Menu Options.....................................................................................3-6
Main Menu Option: DNA Editor ............................................................................................3-7
The Edit Key....................................................................................................................3-8
The Copy Key................................................................................................................3-11
The Print Key.................................................................................................................3-12
Main Menu Option: Start Synthesis ....................................................................................3-13
Main Menu Option: Monitor Synthesis ...............................................................................3-16
Instrument Status: Synthesizing....................................................................................3-17
The Holding Menu .........................................................................................................3-19
The Jump Step Menu ....................................................................................................3-22
The Interrupt Menu........................................................................................................3-24
Main Menu Option: Change Bottles ...................................................................................3-29
Menu Description ..........................................................................................................3-29
ii
Applied Biosystems
The Change Bottles Menu Keys....................................................................................3-30
Bottle Usage Data .........................................................................................................3-33
Main Menu Option: Cycle Editor.........................................................................................3-34
Introduction....................................................................................................................3-34
The Cycle Editor Menu..................................................................................................3-35
The Edit Key..................................................................................................................3-35
The Base Specifier Field ...............................................................................................3-37
The Copy Key................................................................................................................3-39
Main Menu Option: Manual Control....................................................................................3-40
The Valve Key ...............................................................................................................3-40
The Function Key ..........................................................................................................3-41
Main Menu Option: Fract Pulse ..........................................................................................3-42
Main Menu Option: Procedure Editor .................................................................................3-42
The Edit Key..................................................................................................................3-43
The Copy Key................................................................................................................3-43
The Print Key.................................................................................................................3-44
Main Menu Option: FXN Editor ..........................................................................................3-44
The Edit Key..................................................................................................................3-45
The Print Key.................................................................................................................3-46
Main Menu Option: Power Fail ...........................................................................................3-47
Main Menu Option: Self Test ..............................................................................................3-48
Main Menu Option: Set Clock.............................................................................................3-51
Main Menu Option: Shut Down ..........................................................................................3-52
Section 4:
Functions, Cycles and Procedures
Introduction...........................................................................................................................4-2
Valves...................................................................................................................................4-2
Functions ..............................................................................................................................4-5
Synthesis Cycle Functions ............................................................................................4-10
Procedure Functions .....................................................................................................4-15
Test Functions...............................................................................................................4-18
Synthesis Cycles ................................................................................................................4-18
More About the .2µM Cycle...........................................................................................4-20
Procedures .........................................................................................................................4-23
The Phosphoramidite Purge Procedure ........................................................................4-23
The Bottle Change Procedure.......................................................................................4-24
The Shut Down Procedure ............................................................................................4-27
The Flow Test Procedure ..............................................................................................4-28
iii
Applied Biosystems
Section 5:
System Description – Hardware
Introduction...........................................................................................................................5-2
The Chemical Delivery System ............................................................................................5-3
Pressure ..........................................................................................................................5-4
Reagent and Solvent Reservoirs.....................................................................................5-4
Delivery Valve Blocks......................................................................................................5-6
Vacuum Assist.................................................................................................................5-9
The Column...................................................................................................................5-10
Waste and Venting ........................................................................................................5-11
The Battery.........................................................................................................................5-12
The Controller.....................................................................................................................5-13
Fuses..................................................................................................................................5-14
Section 6:
Chemistry for Automated DNA Synthesis
Introduction...........................................................................................................................6-2
The Solid Support - CPG......................................................................................................6-5
DNA Synthesis Chemistry Cycle ..........................................................................................6-7
Detritylation .....................................................................................................................6-8
Coupling ........................................................................................................................6-12
Capping .........................................................................................................................6-15
Oxidation .......................................................................................................................6-16
Completion of the Synthesis Cycle.....................................................................................6-18
Manual Deprotection and Cleavage ...................................................................................6-18
Cleavage .......................................................................................................................6-18
Phosphate Deprotection................................................................................................6-18
Base Deprotection.........................................................................................................6-19
Quantitation of the Oligonucleotide ....................................................................................6-20
Storage of the Oligonucleotide ...........................................................................................6-20
Analysis and Purification ....................................................................................................6-21
OPC, Oligonucleotide Purification Cartridge .................................................................6-21
PAGE ............................................................................................................................6-22
HPLC.............................................................................................................................6-24
Alternative Chemistries ......................................................................................................6-24
RNA Synthesis ..............................................................................................................6-24
Hydrogen-Phosphonate Chemistry ...............................................................................6-25
Phosphorothioate DNA..................................................................................................6-26
5' Attachments....................................................................................................................6-27
Fluorescent-dye Linked Sequencing Primers................................................................6-28
iv
Applied Biosystems
References .........................................................................................................................6-30
Appendix A: Functions, Cycles and Procedures
Appendix B: DNA Synthesizer Schematic
DNA Synthesizer Plumbing Diagram
Appendix C: Synthesis Log Sheet
Reagent/Solvent Log Sheet
Appendix D: 391 Illustrated Parts List
Appendix E: Warranty
391 Pre-Installation Manual
Safety First! ......................................................................................................................... E-3
User Attention Words, Material Safety Data Sheets, and Waste Profiles ........................... E-4
User Attention Words ..................................................................................................... E-4
Material Safety Data Sheets (MSDS)............................................................................. E-4
Waste Profiles ................................................................................................................ E-5
Abbreviations, Initializations, and Units ............................................................................... E-6
Introduction.......................................................................................................................... E-7
Chemicals and Accessories ................................................................................................ E-7
Start-Up Chemical Kit..................................................................................................... E-7
Additional Chemicals and Columns................................................................................ E-9
Shipping List................................................................................................................. E-10
User-Supplied Equipment ............................................................................................ E-11
Site Preparation................................................................................................................. E-13
Laboratory Space ......................................................................................................... E-13
Electrical Requirements ............................................................................................... E-14
Power “Quality”............................................................................................................. E-15
Cooling Requirements.................................................................................................. E-16
Argon............................................................................................................................ E-16
v
Applied Biosystems
Ventilation..................................................................................................................... E-16
Liquid Waste Disposal.................................................................................................. E-17
Operator Training at Installation ................................................................................... E-17
Proof of Performance ................................................................................................... E-17
Printer........................................................................................................................... E-17
Preinstallation Checklist .................................................................................................... E-18
Technical Support ............................................................................................................. E-19
Contacting Technical Support ...................................................................................... E-19
To Contact Technical Support by E-Mail...................................................................... E-19
Hours for Telephone Technical Support....................................................................... E-19
To Contact Technical Support by Telephone or Fax.................................................... E-20
To Reach Technical Support Through the Internet ...................................................... E-22
To Obtain Documents on Demand............................................................................... E-23
AB LIMITED WARRANTY ................................................................................................. E-24
vi
Section 1:
Introduction
How To Use This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2
General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-3
How to Get Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-5
User Attentions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-10
Material Safety Data Sheets (MSDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-10
Text Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-11
Applied Biosystems
How To Use This Manual
This Users Manual is divided into 6 sections; Introduction (1), Operation (2), Software Menu Descriptions (3), Functions, Cycles and Procedures (4), System Description – Hardware (5), and
Chemistry for Automated DNA Synthesis (6). In addition, there are five appendices (refer to the
Table of Contents).
For an overview and a general understanding of the synthesizer, read the General Introduction in
this section and the introductions to Sections 3 through 6. Be sure to read the rest of this section for
other important information such as how to get help if you have questions or problems.
Section 2: Operation, provides step-by-step instructions on how to operate the instrument and perform necessary procedures. Prior to your initial synthesis be sure to read the following parts of this
section: Power On, Pre-synthesis Check List, How to Prepare Phosphoramidites, How to Install
Reagent Bottles, How to Begin a Synthesis, Post Synthesis, and Manual Deprotection and Cleavage. The remaining parts of this section should be read as needed.
Section 3: Software Menu Descriptions, explains how to use each menu option shown on the display screen. A summary of all options appears on page 3-6. Prior to your initial synthesis, be sure
to read the Main Menu Option: DNA Editor, Main Menu Option: Start Synthesis, Main Menu
Option: Monitor Synthesis and Main Menu Option: Change Bottles. Read about the remaining
menu options as you need to use them.
Sections 4, 5 and 6 contain important information about how the instrument operates. It is very useful to read through these sections, but they can also be used as a reference guide. Refer to the Table
of Contents to locate a specific topic. Each section is further described below.
Section 4: Functions, Cycles and Procedures, contains information about valves, functions, synthesis cycles and procedures (e.g., the bottle change procedure). Pages 4-5 and 4-6 describe functions
and show an example of how to use the DNA Synthesizer Schematic. Pages 4-18 and 4-19 describe
the synthesis cycles.
Section 5: System Description – Hardware, describes the components of the synthesizer and explains the chemical delivery system.
Section 6: Chemistry for Automated DNA Synthesis, explains the DNA synthesis chemistry. In addition, it contains an overview of oligonucleotide analysis and purification procedures, a description
of alternative chemistries and 5' attachments, and information about quantifying and storing oligonucleotides.
1-2
Section 1: Introduction
Applied Biosystems
General Introduction
The Applied Biosystems Model 391 DNA Synthesizer automatically performs all steps for DNA
synthesis to produce the highest quality oligonucleotides possible. When used as a system-including AB reagents and columns-the PCR-MATE EP delivers high reliability, ease of operation and
efficient use of your time.
The Model 391 uses the phosphoramidite method of oligonucleotide synthesis because of its inherently high coupling efficiency and the stability of the starting materials. The synthesis begins with
the 3' terminal nucleoside attached to a solid support which is contained within a column (the reaction chamber). Nucleotide 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. Typically, stepwise yields
of 98 to 100% are obtained enabling synthesis of oligonucleotides more than 180 bases in length.
With these high coupling efficiencies, the desired sequence is so abundant in shorter syntheses (up
to ~30 bases) that you usually do not need to purify the product. A simple desalting is adequate for
most applications. When you need to purify longer oligonucleotides, Applied Biosystems offers
OPC, the Oligonucleotide Purification Cartridge. This fast and easy-to-use cartridge completely desalts and purifies the DNA product in about 15 minutes.
All Applied Biosystems chemicals are pretested to ensure high yield syntheses. Solvents and reagents are packaged in bottles that you attach directly to the synthesizer. All chemicals, except the
phosphoramidites, are ready to use. To prepare the phosphoramidites, you only need to dissolve
them in anhydrous acetonitrile. Each phosphoramidite, column, reagent and solvent manufactured
by Applied Biosystems is unconditionally guaranteed. If you are not completely satisfied by the
product (and it is used prior to any applicable expiration date and under the correct operating conditions), it will be replaced at no charge.
Reagents and solvents are delivered to the column by a pressure-driven chemical delivery system.
The system uses patented zero-dead volume valves which increase reliability, eliminate cross-contamination, and reduce the reagent costs.
The Model 391 has menu-driven software designed for simplicity and ease of operation. A 2-line
screen displays various options and necessary information about the synthesis or status of the instrument. In response, you select an option and give instructions by pressing the appropriate key on
the keyboard.
Automated synthesis uses a cycle, consisting of a series of steps, which completes all chemical reactions needed to couple one base. The cycle is repeated until all bases of the oligonucleotide chain
are added. Applied Biosystems supplies four cycles that have been thoroughly tested and optimized
for use with β-cyanoethyl phosphoramidites. They vary according to how much initial 3' terminal
nucleoside is attached to the support (e.g., .2µM, 1µM, 10µM). You decide which one to use based
on how much final product you need for your experiments.
Section 1: Introduction
1-3
Applied Biosystems
The Extended Programmability (EP) feature allows you to create and edit cycles (as well as procedures and functions) optimized for your own needs. And, you can implement alternative chemistries
such as RNA synthesis and hydrogen phosphonate synthesis to create nucleotide analogs. When using these chemistries, you only need to load the appropriate reagents and customize a cycle. No
hardware changes are needed.
The PCR-MATE EP also includes the computer software program, Syncom™. Syncom electronically transfers DNA sequences from an IBM or compatible personal computer (PC) to your synthesizer. With Syncom you can create a vast library of sequences on a PC, then easily transfer a
sequence to the instrument for synthesis. The program offers the opportunity to link your Model
391 to a network of computers to provide Laboratory Information Management (LIM) capabilities.
Applied Biosystems has been perfecting the science of DNA synthesis on automated instruments
since 1982. The PCR-MATE EP is the latest advance, offering the individual laboratory an instrument system that delivers high quality and reliability at an economical price.
1-4
Section 1: Introduction
Applied Biosystems
How to Get Help
Contacting Technical Support
You can contact Applied Biosystems for technical support by telephone or fax, by e-mail, or
through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of
analysis, and other related documents 24 hours a day. In addition, you can download documents in
PDF format from the Applied Biosystems Web site (please see the section “To Obtain Documents
on Demand” following the telephone information below).
To Contact Technical Support by E-Mail
Contact technical support by e-mail for help in the following product areas:
Product Area
E-mail address
Genetic Analysis (DNA Sequencing)
[email protected]
Sequence Detection Systems and PCR
[email protected]
Protein Sequencing,
Peptide and DNA Synthesis
[email protected]
Biochromatography, PerSeptive DNA, PNA and
Peptide Synthesis systems, CytoFluor®, FMAT™,
Voyager™, and Mariner™ Mass Spectrometers
[email protected]
LC/MS
(Applied Biosystems/MDS Sciex)
[email protected]
or
[email protected]
Chemiluminescence (Tropix)
[email protected]
Hours for Telephone Technical Support
In the United States and Canada, technical support is available at the following times:
Product
Hours
Chemiluminescence
8:30 a.m. to 5:30 p.m. Eastern Time
Framingham support
8:00 a.m. to 6:00 p.m. Eastern Time
All Other Products
5:30 a.m. to 5:00 p.m. Pacific Time
Section 1: Introduction
1-5
Applied Biosystems
To Contact Technical Support by Telephone or Fax
In North America
To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below.
(To open a service call for other support needs, or in case of an emergency, dial 1-800-831-6844
and press 1.)
Product or
Product Area
Telephone
Dial...
Fax
Dial...
ABI PRISM® 3700 DNA Analyzer
1-800-831-6844,
then press 8
1-650-638-5981
DNA Synthesis
1-800-831-6844,
then press 21
1-650-638-5981
Fluorescent DNA Sequencing
1-800-831-6844,
then press 22
1-650-638-5981
Fluorescent Fragment Analysis (includes GeneScan®
applications)
1-800-831-6844,
then press 23
1-650-638-5981
Integrated Thermal Cyclers (ABI PRISM ® 877 and
Catalyst 800 instruments)
1-800-831-6844,
then press 24
1-650-638-5981
ABI PRISM ® 3100 Genetic Analyzer
1-800-831-6844,
then press 26
1-650-638-5981
BioInformatics (includes BioLIMS, BioMerge™, and
SQL GT™ applications)
1-800-831-6844,
then press 25
1-505-982-7690
Peptide Synthesis (433 and 43X Systems)
1-800-831-6844,
then press 31
1-650-638-5981
Protein Sequencing (Procise Protein Sequencing
Systems)
1-800-831-6844,
then press 32
1-650-638-5981
PCR and Sequence Detection
1-800-762-4001,
then press 1 for PCR,
2 for the 7700 or 5700,
6 for the 6700
or dial 1-800-831-6844,
then press 5
1-240-453-4613
Voyager MALDI-TOF Biospectrometry and Mariner
ESI-TOF Mass Spectrometry Workstations
1-800-899-5858,
then press 13
1-508-383-7855
Biochromatography (BioCAD Workstations and
Poros Perfusion Chromatography Products)
1-800-899-5858,
then press 14
1-508-383-7855
Expedite Nucleic acid Synthesis Systems
1-800-899-5858,
then press 15
1-508-383-7855
Peptide Synthesis (Pioneer and 9050 Plus Peptide
Synthesizers)
1-800-899-5858,
then press 15
1-508-383-7855
PNA Custom and Synthesis
1-800-899-5858,
then press 15
1-508-383-7855
1-6
Section 1: Introduction
Applied Biosystems
Product or
Product Area
Telephone
Dial...
Fax
Dial...
FMAT 8100 HTS System and Cytofluor 4000
Fluorescence Plate Reader
1-800-899-5858,
then press 16
1-508-383-7855
Chemiluminescence (Tropix)
1-800-542-2369 (U.S.
1-781-275-8581
only),
or 1-781-271-0045
Applied Biosystems/MDS Sciex
1-800-952-4716
1-650-638-6223
Telephone
Dial...
Fax
Dial...
Outside North America
Region
Africa and the Middle East
Africa (English Speaking) and West Asia (Fairlands,
South Africa)
27 11 478 0411
27 11 478 0349
South Africa (Johannesburg)
27 11 478 0411
27 11 478 0349
Middle Eastern Countries and North Africa (Monza,
Italia)
39 (0)39 8389 481
39 (0)39 8389 493
Eastern Asia, China, Oceania
Australia (Scoresby, Victoria)
61 3 9730 8600
61 3 9730 8799
China (Beijing)
86 10 64106608
86 10 64106617
Hong Kong
852 2756 6928
852 2756 6968
Korea (Seoul)
82 2 593 6470/6471
82 2 593 6472
Malaysia (Petaling Jaya)
60 3 758 8268
60 3 754 9043
Singapore
65 896 2168
65 896 2147
Taiwan (Taipei Hsien)
886 2 22358 2838
886 2 2358 2839
Thailand (Bangkok)
66 2 719 6405
66 2 319 9788
Europe
Austria (Wien)
43 (0)1 867 35 75 0
43 (0)1 867 35 75 11
Belgium
32 (0)2 712 5555
32 (0)2 712 5516
Czech Republic and Slovakia (Praha)
420 2 61 222 164
420 2 61 222 168
Denmark (Naerum)
45 45 58 60 00
45 45 58 60 01
Finland (Espoo)
358 (0)9 251 24 250
358 (0)9 251 24 243
France (Paris)
33 (0)1 69 59 85 85
33 (0)1 69 59 85 00
Germany (Weiterstadt)
49 (0) 6150 101 0
49 (0) 6150 101 101
Hungary (Budapest)
36 (0)1 270 8398
36 (0)1 270 8288
Italy (Milano)
39 (0)39 83891
39 (0)39 838 9492
Norway (Oslo)
47 23 12 06 05
47 23 12 05 75
Poland, Lithuania, Latvia, and Estonia (Warszawa)
48 (22) 866 40 10
48 (22) 866 40 20
Portugal (Lisboa)
351 (0)22 605 33 14
351 (0)22 605 33 15
Section 1: Introduction
1-7
Applied Biosystems
Region
Telephone
Dial...
Fax
Dial...
Russia (Moskva)
7 095 935 8888
7 095 564 8787
South East Europe (Zagreb, Croatia)
385 1 34 91 927
385 1 34 91 840
Spain (Tres Cantos)
34 (0)91 806 1210
34 (0)91 806 1206
Sweden (Stockholm)
46 (0)8 619 4400
46 (0)8 619 4401
Switzerland (Rotkreuz)
41 (0)41 799 7777
41 (0)41 790 0676
The Netherlands (Nieuwerkerk a/d IJssel)
31 (0)180 331400
31 (0)180 331409
United Kingdom (Warrington, Cheshire)
44 (0)1925 825650
44 (0)1925 282502
All other countries not listed
(Warrington, UK)
44 (0)1925 282481
44 (0)1925 282509
81 3 5566 6230
81 3 5566 6507
Japan
Japan (Hacchobori, Chuo-Ku, Tokyo)
Latin America
Del.A. Obregon, Mexico
305-670-4350
305-670-4349
To Reach Technical Support Through the Internet
We strongly encourage you to visit our Web site for answers to frequently asked questions and for
more information about our products. You can also order technical documents or an index of available documents and have them faxed or e-mailed to you through our site. The Applied Biosystems
Web site address is
http://www.appliedbiosystems.com/techsupp
To submit technical questions from North America or Europe:
Step
Action
1
Access the Applied Biosystems Technical Support Web site.
2
Under the Troubleshooting heading, click Support Request Forms, then select the relevant
support region for the product area of interest.
3
Enter the requested information and your question in the displayed form, then click Ask Us RIGHT
NOW (blue button with yellow text).
4
Enter the required information in the next form (if you have not already done so), then click Ask
Us RIGHT NOW.
You will receive an e-mail reply to your question from one of our technical experts within 24 to 48
hours.
1-8
Section 1: Introduction
Applied Biosystems
To Obtain Documents on Demand
Free, 24-hour access to Applied Biosystems technical documents, including MSDSs, is available
by fax or e-mail or by download from our Web site.
To order
documents...
Then...
by index number
a. Access the Applied Biosystems Technical Support Web site at
http://www.appliedbiosystems.com/techsupp
b. Click the Index link for the document type you want, then find the document you
want and record the index number.
c. Use the index number when requesting documents following the procedures
below.
by phone for fax
delivery
a. From the U.S. or Canada, call 1-800-487-6809, or
from outside the U.S. and Canada, call 1-858-712-0317.
b. Follow the voice instructions to order the documents you want.
Note
through the
Internet for fax or
e-mail delivery
There is a limit of five documents per request.
a. Access the Applied Biosystems Technical Support Web site at
http://www.appliedbiosystems.com/techsupp
b. Under Resource Libraries, click the type of document you want.
c. Enter or select the requested information in the displayed form, then click Search.
d. In the displayed search results, select a check box for the method of delivery for
each document that matches your criteria, then click Deliver Selected Documents
Now (or click the PDF icon for the document to download it immediately).
e. Fill in the information form (if you have not previously done so), then click Deliver
Selected Documents Now to submit your order.
Note There is a limit of five documents per request for fax delivery but no limit on
the number of documents you can order for e-mail delivery.
Section 1: Introduction
1-9
Applied Biosystems
User Attentions
Four user-attention words appear in the text of this manual. Each one implies a certain level of observation or action as follows:
Note
This word is used to call attention to information.
IMPORTANT This word is used when information is necessary for proper instrument operation.
Caution
This word informs you that damage to the instrument could result if you do not
comply with this information.
WARNING
Physical injury to you or other people could result if these required
precautions are not taken.
Material Safety Data Sheets (MSDS)
WARNING
1-10
Some chemicals used with this instrument are considered hazardous. Hazards
are prominently displayed on the labels of all hazardous chemicals. In addition,
there are MSDS which provide information about physical characteristics,
hazards, precautions, first aid, spill cleanup and disposal procedures. The
MSDS appear in the User’s Safety Information Section in the front of this
manual. Please be sure to familiarize yourself with the information in these
documents before attempting to operate the instrument or use the reagents.
Additional copies of the MSDS are available from Applied Biosystems at no
extra cost.
Section 1: Introduction
Applied Biosystems
Text Conventions
Menu displays and keys appear in this manual as follows:
1. Menus are boxed and appear centered on the page:
DNA
start
change
cycle
more
editor
synth
bottles
editor
menu
2. When describing what a soft key does, the key is boxed and appears at the left margin:
DNA
EDITOR
Creates, edits and prints up to four DNA sequences
3. When the text refers to a key that you will press, the key appears in capital letters:
When you select DNA EDITOR, the screen displays one of the DNA strands and shows the
number of bases in that strand.
Section 1: Introduction
1-11
Section 2:
Operation
This section describes step-by-step procedures for operating the Model 391 DNA Synthesizer. If
questions arise about the software that are not explained in this section, refer to Section 3 which
describes all menus and keys in detail. Before beginning a synthesis, be sure to read the following
parts: Pre-synthesis Check List, How to Prepare Phosphoramidites, How to Install Reagent Bottles,
How to Begin a Synthesis, Post Synthesis, Manual Deprotection and Cleavage, and How to Store
Reagents. Use the rest of the section as you need to.
Power On. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2
Pre-synthesis Check List. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
How to Prepare Phosphoramidites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4
How to Dissolve Phosphoramidites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5
How to Store Dissolved Phosphoramidites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6
How to Install Reagent Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7
How to Begin a Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9
How to Install the Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-12
Post Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13
Manual Deprotection and Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13
How to Abort a Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-18
About the Synthesis Reagents and Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-19
How to Store Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-19
About Deoxyinosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-23
How to Change an Argon Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-25
How to Change the Waste Bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-25
How to Perform the Flow Test Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-26
Aminolink 2™ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-30
How to Use Aminolink 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-32
How to Use OPC (Oligonucleotide Purification Cartridge) . . . . . . . . . . . . . . . . . . . . . . . . .2-35
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-40
Applied Biosystems
Power On
1. Connect the instrument to a power source and turn the power switch on. The power switch is
located on the front of the instrument to the right of bottle position 5.
IMPORTANT
The argon cylinder must always be connected to the synthesizer before
the power is turned on. If this is not done, the vacuum-assist valves will
be activated continuously and can overheat and fail causing argon to
leak.
2. Select START from the following display:
Applied Biosystems 391 DNA Synthesizer
PCR-MATE EP
Ver. 1.00
start
Page 1 of the Main Menu will then be shown:
DNA
start
change
cycle
more
editor
synth
bottles
editor
menu
The display can be adjusted for optimal viewing by turning the knob at the left of the screen.
2-2
Section 2: Operation
Applied Biosystems
Pre-synthesis Check List
Before beginning a synthesis, be sure to complete the following steps.
1. Check reagent levels of all reservoirs.
Empty reservoirs should be replaced with bottles of fresh reagents. From the Main Menu,
choose CHANGE BOTTLES and follow the prompts. For further instructions, refer to How
to Install Reagent Bottles found later in this section.
Be sure bottles are on every position. Since all phosphoramidite reservoirs are pressurize
simultaneously with a single valve, all 5 bottles must be attached to the instrument (even if
some are empty) to perform synthesis.
2. Check that all alarms are set correctly. Refer to Section 3: Main Menu Option: Change Bottles,
for instructions on how to set the alarm.
3. As needed, prepare the phosphoramidites for use by dissolving them in anhydrous
acetonitrile. For further instructions, refer to How to Prepare Phosphoramidites later in this
section.
4. Check the pressure supply of the argon tank. Check that the low pressure gauge reads 60 psi.
Change the tank as it is depleted. Watch it carefully when the high pressure gauge drops below
400 psi. With average synthesizer use, an argon tank should last approximately 3 months. To
change an argon tank, refer to the instructions later in this section.
5. Check the waste level.
The synthesizer generates 1 to 2 liters of hazardous, halogenated, organic liquid waste per 100
base additions. The waste is collected in a 4-liter bottle supplied by Applied Biosystems.
When the waste bottle is full, it must be emptied and the waste disposed of properly. The bottle
can be changed prior to a synthesis or when a synthesis is interrupted. To change the waste
bottle, refer to the instructions later in this section.
6. Fill the fraction collector with 10- to 15-mL volumetric tubes to collect the trityl cation
released at each detritylation step. Use one tube for each base in the sequence. Be sure to label
the tubes and align the fraction collector so tube number one collects the first trityl.
Section 2: Operation
2-3
Applied Biosystems
How to Prepare Phosphoramidites
The prepackaged phosphoramidites are bottled as powders and sealed under argon pressure. In this
state, they are stable for at least 1 year. To prepare them for use, they are dissolved in anhydrous
acetonitrile.
Since the phosphoramidites are extremely sensitive to acid, oxygen, and water, you must take special care when dissolving them. The following instructions will help avoid contamination, prevent
degradation and ensure high coupling yields.
Important factors to consider when dissolving phosphoramidites:
1. Use anhydrous acetonitrile with less than 90 ppm water to dissolve the phosphoramidites. Use
of Applied Biosystems anhydrous acetonitrile, Part Number 400060, is strongly
recommended. Do not use HPLC grade acetonitrile, its higher water content will decrease
coupling efficiency. After opening the acetonitrile, keep it blanketed with argon to avoid
contamination with air.
2. When transferring the acetonitrile to a phosphoramidite bottle, use a clean, dry, glass syringe
with a needle. Store it in a 100-120°C oven to prevent atmospheric moisture contamination.
Keep the syringe dedicated to this use. Use acetonitrile to rinse it, do not use water. Do not
contaminate the acetonitrile bottle with traces of phosphoramidites (i.e., do not allow the
syringe needle to contact the phosphoramidites).
3. Add the correct amount of acetonitrile to each phosphoramidite as follows:
β-cyanoethyl
phosphoramidite
A
G
C
T
Deoxyinosine
volume of
acetonitrile
(mL)
2.8
5.6
11.2
2.9
5.8
11.6
2.9
5.9
11.8
3.3
6.6
13.2
3.4
weight of
phosphoramidite
(grams)
.25
.5
1.0
.25
.5
1.0
.25
.5
1.0
.25
.5
1.0
.25
molarity
(M)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
The above concentrations result in a 10-fold excess of soluble phosphoramidite over support-bound
nucleoside.
2-4
Section 2: Operation
Applied Biosystems
How to Dissolve Phosphoramidites
1. To prepare the phosphoramidite bottle, pull back the aluminum tab in the direction of the
arrow. Do not yet remove it, simply expose the septum. Place a needle (any gauge, without a
syringe) into the rubber septum. This vents the pressure in the bottle when the anhydrous
acetonitrile is added. Venting also prevents accidental splashing when the phosphoramidite
bottle is opened and placed on the instrument.
2. Unscrew the cap from the anhydrous acetonitrile bottle and quickly replace it with a clean
rubber septum. The acetonitrile is bottled under argon. Since argon is heavier than air, argon
should still blanket the acetonitrile after the septum transfer.
3. Remove the syringe/needle from the oven and allow it to cool to room temperature. Pierce the
septum of the acetonitrile bottle with the needle and remove the correct amount of acetonitrile.
4. Pierce the septum of the phosphoramidite bottle a few millimeters with the needle/syringe and
slowly add the acetonitrile. Make sure the needle does not touch the phosphoramidite powder
or solution. When finished, remove both the venting needle and the needle/syringe and gently
swirl the bottle to dissolve the phosphoramidites. Once dissolved, you can place them on the
instrument. Use the Change Bottles Menu and follow the prompts (see Section 3 for details).
Also, refer to How to Install Reagent Bottles, for instructions on attaching the bottles to the
instrument.
5. The anhydrous acetonitrile bottle must now be blanketed with argon. To do so, place a needle
on the upper male Luer fitting on the front of the synthesizer (remove the column if
necessary). Select MANUAL CONTROL from the Main Menu and activate function 2, a
reverse flush. Argon will then flow out of the needle. Next, push the acetonitrile bottle onto
the needle so that the needle pierces the bottle’s septum. Hold it for 5 seconds and then remove
the bottle and the needle simultaneously. The pressure will immediately be released through
the needle but the bottle will remain blanketed with argon. Remove the needle from the bottle
and continue dissolving additional phosphoramidites as necessary.
Once in solution, phosphoramidites are stable for about two weeks. After this time, coupling efficiencies may slowly begin to decrease. If phosphoramidites cannot be used within this time, it is
possible to freeze, store, thaw and reuse them. Although the stored phosphoramidites may show
some slight loss of activity, they should be adequate for synthesizing sequences of approximately
20 bases.
Section 2: Operation
2-5
Applied Biosystems
How to Store Dissolved Phosphoramidites
If you cannot use the phosphoramidites within about two weeks, you can remove them from the instrument, store them and reuse them later. Do not use the Change Bottles Procedure for this. Instead
follow the protocol below.
1. Using MANUAL CONTROL, activate the appropriate functions, F62 to F66, for 10 seconds,
then deactivate them.
Argon to Reservoirs
F 62 Flush to A
F 63 Flush to G
F 64 Flush to C
F 65 Flush to T
F 66 Flush to X
2. Remove the bottles (without using the change bottles procedure) and quickly cap with
previously unused rubber septa (Aldrich catalog number Z10,074-9).
3. Rinse the delivery lines with acetonitrile by activating the appropriate functions, F71 to F75,
for 10 seconds. Flush the lines with argon by activating the appropriate functions, F62 to F66,
for 10 seconds.
Acetonitrile to
Reservoirs
F 71 #18 to A
F 72 #18 to G
F 73 #18 to G
F 74 #18 to T
F 75 #18 to X
Argon to
Reservoirs
F 62 Flush to A
F 63 Flush to G
F 64 Flush to C
F 65 Flush to T
F 66 Flush to X
4. Seal the bottles with Paraffin film, place in a desiccator containing Drierite and put in a freezer
at -20°C.
5. When ready to synthesize, thaw the phosphoramidites, remove them from the desiccator and
place them on the instrument using the Change Bottles Menu. Be sure to enter an appropriate
alarm setting.
2-6
Section 2: Operation
Applied Biosystems
How to Install Reagent Bottles
A reagent bottle can be changed before beginning a synthesis or when an active synthesis has been
stopped by setting an interrupt or by the alarm. When removing and replacing bottles, always select
CHANGE BOTTLES from page 1 of the Main Menu and follow the prompts. An exception is if
you are going to store and later reuse the phosphoramidites (refer to How to Store Dissolved Phosphoramidites for details).
WARNING
Consider each chemical in the synthesizer potentially harmful. Do not inhale
vapors. Work in a well ventilated area. Always use eye protection and wear
acid-impermeable gloves and a lab coat. Do not leave any chemicals
uncapped. If any chemical is ingested or comes in contact with the eyes,
immediately consult a physician. If there is any physical contact, wash
immediately with ample water. Refer to About the Synthesis Reagents and
Solvents and the MSDS for further instructions about storing and handling
each reagent.
Note
The phosphoramidites, tetrazole and acetonitrile are atmosphere sensitive. Upon
opening one of these bottles, quickly place it on the instrument to prevent
contamination.
How to Install the Phosphoramidites (bottles 1-5)
These reservoirs are push-on bottles that fit around a Teflon insert and silicone O-ring to form an
airtight seal inside each bottle neck. Pressing the appropriate black button above the bottle position
releases the grip on the bottle and allows for its removal and subsequent replacement. When installing a bottle, the button will return to its out position only when the reservoir is correctly engaged.
1. To remove a bottle, firmly pull it straight down while pressing the black button above its
receptacle. If the bottle seems to stick, carefully move it side to side while pulling it off. Cover
or recap the bottle immediately to minimize vapor release. Wipe the delivery line with a lintfree tissue.
2. Dissolve the phosphoramidites as described in How to Prepare Phosphoramidites found in this
section. Next, remove the aluminum cap and the rubber septum from the bottle. Wipe any
crystals or drops of reagent off the bottle neck.
3. To install the bottle, firmly push it up around its receptacle while pressing the black button.
As necessary, maneuver the bottle into place by carefully moving it side to side while pushing.
4. When the bottle is correctly engaged, release the button and it will return to its out position.
If the button remains in, the bottle is not seated properly and must be repositioned.
Section 2: Operation
2-7
Applied Biosystems
How to Install Bottles 9-15
These reservoirs screw directly into a threaded cap mounted on the synthesizer.
1. To remove a bottle, unscrew it by turning clockwise. Remove the disposable polyethylene
insert. Recap it immediately to minimize vapor release.
Note
The disposable insert forms an airtight seal between each cap assembly and
bottle. It is designed for single use and should be replaced with each bottle
change. Inserts are supplied at no charge when you order the chemical reagent
kits. To order inserts separately, use the following Part Numbers (PN): for
450mL bottles, 400501; for 200mL bottles, 400790.
2. Open the full bottle. Place a new polyethylene insert inside the bottle neck. Screw the bottle
snugly into its threaded cap on the instrument by turning it counterclockwise.
Note
Receptacles 9-15 have a ratchet cap assembly. When attaching these bottles,
you cannot overtighten them.
How to Install Bottle 18
Reservoir 18, acetonitrile, is a 4-Liter bottle that is placed on the left side of the instrument, inside
a protective carrier and metal rack. To replace the bottle, slowly unscrew the cap assembly to release pressure. Remove it and then screw it on a fresh acetonitrile bottle.
IMPORTANT Use HPLC or UV grade acetonitrile with a specification of less than 300 ppm of water.
Water contamination of even 1000 ppm will lead to a 1 to 2% decrease in coupling
efficiency.
2-8
Section 2: Operation
Applied Biosystems
How to Begin a Synthesis
After completing the pre-synthesis check list, perform the following steps to begin a synthesis. As
questions arise, refer to Section 3: Software Menu Descriptions, for further instructions and explanations.
1. View Page 1 of the Main Menu:
DNA
start
change
cycle
more
editor
synth
bottles
editor
menu
DNA-1
0 mer
next
main
2. From the Main Menu, select DNA EDITOR:
Select action for
edit
copy
print
Press NEXT until the desired DNA strand is in view, then press EDIT:
5'>_
<3' 0
(
space
)
erase
menu
Enter the sequence (5' to 3') using the A, G, C, T and X keys. Be sure the sequence is correct. If
possible, print and verify it. When finished, return to the Main Menu by selecting MENU and then
MAIN.
Note that any combination of phosphoramidites can be inserted in any position, except the 3'-terminus, for synthesizing mixed-sequence probes. This is done by pressing the left, open parenthesis,
( , typing the desired base keys, and pressing the right, close parenthesis, ). Multiple bases are added
to the support-bound nucleoside by simultaneous delivery of the specified phosphoramidites.
3. From the Main Menu, select START SYNTH:
Make
DNA-1:12
Cycle-1:63
begin
nxt DNA
nxt cyc
Trityl OFF
ON/OFF
main
Configure the synthesis.
a. Press NXT DNA until the correct DNA strand to be synthesized is showing. The most
recently edited strand will automatically appear. The above example shows DNA-1 which
is a 12 base sequence.
b. Press NXT CYC until the desired synthesis cycle is shown. When the main power is first
turned on, all Applied Biosystems ROM cycles are automatically loaded into the correct
RAM cycle location as shown below. For example, to synthesize using the .2µM cycle,
Section 2: Operation
2-9
Applied Biosystems
Cycle-1:63 should be displayed. (You can create your own cycles by editing a RAM cycle.
Once you change a RAM cycle, the edited version will appear in the Start Synthesis Menu.
You can, however, copy a ROM cycle back into a RAM cycle location at any time. Refer
to Section 3: Main Menu Option: Cycle Editor, for instructions.)
ROM
synthesis
cycle
RAM cycle
location
Total
number of
steps
cycle time
(min.)
crude
yield* (O.D.)
(20mer)
.2µM
Cycle-1
63
5.5
20-25
1µM
Cycle-2
64
5.5
100-120
Low**
Cycle-3
61
5.5
20-25
10µM
Cycle-4
53
25
800-1000
*Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming
33 micrograms/O.D. unit. **The Low cycle is on the .2µM scale.
The synthesis cycle, Low, is a low-reagent consumption cycle on the .2µM scale. It uses
33% less phosphoramidites than the .2µM cycle, although its coupling efficiencies are
slightly lower, about 97%. “Low” can be used to synthesize sequences less than 50 bases.
Sequences less than 30 bases usually only need desalting. Sequences greater that 30 bases
should be OPC purified.
c. Press ON/OFF until the desired ending method is shown.
The 5' terminus of the fully synthesized DNA chain can either remain protected by a
dimethoxytrityl group (trityl on), or can be detritylated to yield a 5'-hydroxyl (trityl off).
Trityl on is usually selected when purifying by trityl-specific OPC or reverse phase HPLC.
Trityl off is usually chosen when purifying by gel electrophoresis or ion exchange HPLC.
When all information is displayed correctly, select BEGIN. All bottles will then be checked for usage:
Checking bottle usage for base 1
Please Wait . . .
2-10
Section 2: Operation
Applied Biosystems
According to the alarm you set in the Change Bottles Menu, a message will be displayed if the alarm
will be triggered during the synthesis. For example, if bottle 9, tetrazole, will empty in 11 cycles,
the screen will read:
Bottle 9 will interrupt in 11 cycles
begin
menu
Check the reagent level of the bottle. Replace it with fresh reagent, if necessary. To do this, return
to the Main Menu, select CHANGE BOTTLES and follow the instructions. If the bottle is not empty, you can reenter an appropriate alarm setting in the Change Bottles Menu. Check the reagent level
of the other bottles and decide whether to replace them now or wait until the synthesis is interrupted
by the alarm.
When ready to start synthesis, press BEGIN. The screen will show a display similar to:
Install (T) COLUMN, then press enter
enter
menu
Place the correct color-coded column on the instrument. This column will correspond to the base at
the 3' end of your DNA sequence. Make sure it matches what is displayed on the screen and is the
correct synthesis scale. Record the column’s serial number to identify the synthesis.
CPG-bound
nucleoside
A
G
C
T
column
color-code
green
yellow
red
blue
Small scale (.2µM) columns have labels with broken colored lines and serial numbers that begin
with “2”. One-micromole columns have labels with solid colored lines and serial numbers that begin with “3”. Ten-micromole columns are larger than the others. They have labels with solid colored
lines and serial numbers that begin with “3”.
Wide-pore CPG columns are recommended when synthesizing oligonucleotides greater than 80
bases. They are available on the .2µM scale only. Wide-pore columns have labels with broken colored lines and serial numbers that begin with “4”.
Section 2: Operation
2-11
Applied Biosystems
How to Install the Column
1. Remove the aluminum tabs from both ends and tap the ends on a dark surface to check for
leaks. If CPG falls out, do not use the column. Any faulty columns will be replaced by Applied
Biosystems without charge.
2. Firmly push either end straight up onto the upper male Luer fitting on the instrument. Since
the column is symmetrical, it can be attached in any way.
3. Firmly push the lower male Luer fitting straight up into the bottom of the column. The column
should fit securely. Do not twist the fittings or the filters may crimp or tear.
Once the column is installed, select ENTER. The screen will read:
Purge Phosphoramidite lines?
no
yes
menu
Choose whether to purge the phosphoramidite and tetrazole delivery lines. Press YES if the instrument has been idle for more than 12 hours (more than 6 hours in humid environments) or if a phosphoramidite reservoir has not been accessed within 12 hours.
Press NO if the instrument has been running continuously and if all the phosphoramidites have been
accessed within 12 hours. Upon pressing NO or after the purge is finished, the synthesis automatically begins and page one of the Synthesizing Status Menu (one of the Monitor Synthesis Menus)
is shown:
SYNTHESIZING base 1 of xx bases
hold
jump
intrpt
more
main
In the above menu, “xx” equals the total number of bases in the cycle. Press the MORE key to view
the current synthesis cycle step. Press MORE again to view the DNA sequence and the base currently being synthesized. Press MORE again to view the synthesis configuration. From this menu,
you can hold or interrupt a step, or jump to another step in the cycle. Refer to Section 3: Main Menu
Option: Monitor Synthesis, for details on how to hold, interrupt and jump a step.
Notes for after synthesis begins:
Following synthesis start, check that the column does not leak during chemical deliveries. Also,
note if and when bottles will need replacing.
During the synthesis, you can continue to view the Monitor Synthesis Menu or return to the Main
Menu to access other ones. During an active synthesis, Self Test, Manual Control and Shut Down
cannot be accessed. However, you can access the Cycle Editor during synthesis to change cycle step
times.
2-12
Section 2: Operation
Applied Biosystems
When the synthesis is finished, SYNTHESIS COMPLETE is displayed in the Monitor Synthesis
Menu.
Post Synthesis
1. Remove the column. To identify it, be sure to record its serial number.
2. Remove the trityl tubes. Visually inspect the trityl solutions. Be sure there are no clear tubes
and that the intensity of the orange color gradually decreases. Perform the trityl cation assay
to determine the coupling efficiency of the synthesis. Refer to User Bulletin 13 (Revised),
Evaluation and Purification of Synthetic Oligonucleotides, for instructions. Once the column
and trityl tubes are removed, the instrument is ready to begin another synthesis.
3. Perform the Manual Deprotection and Cleavage Procedure described on the next page.
4. If desired, analyze the crude oligonucleotide mixture by one of the methods, such as HPLC
or gel electrophoresis, described in User Bulletin 13 (Revised).
5. Prepare the DNA for use in experiments by removing the ammonia and then desalting. Purify
the product, if necessary. Applied Biosystems Oligonucleotide Purification Cartridge (OPC)
provides a fast and easy way to purify and desalt in one step. An overview of OPC, and other
purification methods, can be found in Chemistry for Automated DNA Synthesis. In addition,
refer to How to Use OPC (Oligonucleotide Purification Cartridge) found later in this section.
Refer to User Bulletin 13 (Revised) for other detailed instructions about analysis and
purification.
Manual Deprotection and Cleavage
Introduction
After the synthesis of a deoxyoligonucleotide on the Model 391, the DNA produced is not yet biologically active. The phosphate groups and base exocyclic amines are protected to prevent side reactions during synthesis. Following synthesis, the protecting groups must be removed. In addition,
the DNA must be hydrolyzed or cleaved from the solid support.
There are three types of protecting groups:
1. 5' protecting group - dimethoxytrityl (DMT)
2. β-cyanoethyl phosphate protecting groups
3. base protecting groups - benzoyl on dA and dC and isobutyryl on dG
The 5'-protecting group, dimethoxytrityl, is attached to each nucleoside phosphoramidite and is
cleaved from the growing oligonucleotide chain during each cycle of base addition. You can program the 391 to remove the last DMT to yield a 5' hydroxyl by choosing the ending method Trityl
off, or leave the DMT on by choosing the ending method Trityl on. When purifying by polyacrylaSection 2: Operation
2-13
Applied Biosystems
mide gel electrophoresis (PAGE)1 or ion exchange HPLC2, the last DMT group should be removed.
When purifying by trityl-specific OPC or reverse phase HPLC, the last DMT group should be left
on.1
After the synthesis is complete (either Trityl on or Trityl off), remove the column from the instrument. The oligonucleotides are then simultaneously decyanoethylated and cleaved from the support
using concentrated ammonium hydroxide. Next, the base protecting groups are removed by the addition of fresh concentrated ammonia and incubation at 55ºC.
Materials and Methods
The recommended method for manual deprotection and cleavage was developed by Tanaka and
Letzinger3 and later adapted for use with Applied Biosystems synthesis columns by Schott.4 The
procedure presented here is similar to Dr. Schott’s with minor variations.
Reagents, solvents and apparatus
The manual deprotection kit (Part Number 400257) accompanying the installation kit contains the
following items for the deprotection.
1. Ten 1-mL disposable syringes with Luer fittings (VWR Catalog #BD5602).
2. Ten disposable needles with female Luers (VWR Catalog #BD5167).
3. A number 10 rubber stopper (VWR Catalog #59580-386).
4. Five male-to-male Luer connectors (Alltech Associates Inc. Catalog #86506).
Items 1 and 2 are used on a one-time basis and should be ordered in sufficient quantities for regular
use. Because it is convenient to perform the deprotection on several oligonucleotides simultaneously, you should purchase more male-to-male Luer connectors.
Reagent grade, concentrated ammonium hydroxide is provided in the installation reagent kit. Additional quantities should be purchased from a local supplier. Small DNA collection vials with a
rubber-lined screw cap are available from Applied Biosystems (Part No. 400048) or can be purchased from a local source. However, use Teflon™-lined caps with the vials because the rubberlined caps can leach contaminants into the DNA-ammonium hydroxide solution. They can be ordered from Wheaton; Part Number 240408, size 13-425.
2-14
Section 2: Operation
Applied Biosystems
Figure 2-1.
Manual Deprotection Apparatus
Section 2: Operation
2-15
Applied Biosystems
Methods
1. After the synthesis is complete, remove the column from the instrument and attach one end to
the syringe Luer. Connect the male-to-male Luer to the other end of the column. Next, attach
the syringe needle to the male-to-male Luer. See Figure 2-1.
2. With the syringe plunger inserted into the syringe barrel to the 0.5 mL mark, insert the needle
into some fresh, room temperature ammonium hydroxide. Draw up enough reagent to fill the
column but minimize the volume in the syringe.
IMPORTANT
The concentration of ammonia is critical. Use fresh, concentrated
ammonium hydroxide which has been opened less than one month.
3. Insert the needle into the rubber stopper and let stand for 15-30 minutes. This treatment begins
cleavage and cyanoethyl deprotection.
4. Expel the ammonia into a small DNA collection vial. There can be positive pressure inside
the syringe system which can force some of the ammonia solution out of the needle as it is
removed from the stopper. This can be minimized by using ammonium hydroxide at room
temperature and by slightly withdrawing the syringe barrel as you remove the needle from the
stopper.
IMPORTANT
Use a tightly sealed DNA collection vial that can withstand positive
pressure. The vial must also have a Teflon-lined cap. Rubber-lined
caps have contaminants that leach out of the cap liner during base
deprotection. Teflon-lined caps can be ordered from Wheaton; Part
Number 240408, size 13-425.
5. Repeat the ammonia treatment 3 more times for 15 to 30 minutes each. This ensures complete
cleavage and cyanoethyl deprotection.
IMPORTANT
Remember that the product DNA is now in solution and no longer
bound to the support. Save the column until the cleavage is confirmed.
6. To remove the exocyclic amine base protecting groups (benzoyl and isobutyryl), first bring
the volume of the crude DNA solution to 3mL with fresh concentrated ammonium hydroxide.
Then place the vial of DNA at 55ºC for 8 to 15 hours. Longer treatment is advisable if the
ammonium concentration is questionable. This also cleaves the acetyl caps from the failure
sequences.
7. After completing deprotection, cool the ammonium hydroxide-DNA solution.
a. When purifying by OPC, the oligonucleotide solution is diluted 1:3 with water and then
loaded directly on the cartridge with no other preparation needed. For details, refer to How
to Use OPC (Oligonucleotide Purification Cartridge) found later in this section.
2-16
Section 2: Operation
Applied Biosystems
b. If the DMT group was removed previously as a part of the synthesis cycle, the DNA is
ready for analysis and/or purification by PAGE or ion exchange HPLC. Analysis and
purification procedures are discussed in User Bulletin 13 (Revised).1
c. When purifying by trityl-specific reverse phase HPLC, cool the ammonium hydroxideDNA solution (to prevent losses from bubbling) and remove the ammonia by vacuum.
Keep the solution basic to prevent accidental detritylation by adding one drop of distilled
triethylamine every 10 minutes. Also, avoid heating the sample.
After collection and concentration of the product from reverse phase HPLC, detritylate the
dried sample by dissolving it in 200-500 µL of 80% acetic acid. Since the acetic acid is an
aqueous solution, the trityl cation will react with water and form tritanol and will not give
an orange color. After 20 minutes, add an equal volume of 95% ethanol, and lyophilize the
sample. The dried sample is then lyophilized from ethanol until no acetic acid remains.
The hydrolyzed DMT group can be removed by methods discussed in User Bulletin 13
(Revised).1
Section 2: Operation
2-17
Applied Biosystems
How to Abort a Synthesis
When a synthesis is aborted, all valves close and you will not be able to continue the run. A procedure for how to abort a synthesis appears below. If you need further details, refer to The Interrupt
Menu in Section 3.
Caution
Interrupt synthesis at a safe step, such as Cycle Entry or Cycle End. Otherwise,
chemicals could be left in valve blocks and delivery lines. Salts could then
form preventing subsequent deliveries. The resulting repairs are expensive.
To abort synthesis:
1. Press INTRPT from the Monitor Synthesis Menu.
2. From the No Interrupt Set page of the Interrupt Menu, press AHEAD. Set an Interrupt Ahead
to occur at the next Cycle Entry or Cycle End step. Refer to table below to determine the exact
step number. To interrupt at the next base, do not enter a base number. (Note that if an Interrupt
Ahead has already been set, you can change it by typing over existing entries.)
Synthesis
Cycle
.2µM
1µM
Low
10µM
Cycle Entry
step number
36
36
34
37
Cycle End
step number
63
64
61
53
3. When the synthesis interrupts, the screen will display the Interrupted Status. Press ABORT.
A menu confirming your action appears.
4. Press YES to abort the synthesis. Once you choose YES, the synthesis cannot be resumed.
If you abort synthesis at a step other than Cycle Entry or Cycle End, the lines and valve blocks must
be rinsed and dried. To do this, select Manual Control and activate the following functions for 10
seconds each:
Function 10, “#18 TO WASTE”
Function 9, “#18 TO COLUMN”
Function 1, “BLOCK FLUSH”
Function 2, “REVERSE FLUSH”
2-18
Section 2: Operation
Applied Biosystems
About the Synthesis Reagents and Solvents
All chemicals have been thoroughly tested at Applied Biosystems to ensure repeatedly reliable syntheses. Each reagent and solvent has a specific position on the instrument and is referred to by the
number located above each receptacle. Position numbers are also printed on the bottle labels. The
numbers are 1-5, 9, 11, 12, 14, 15 and 18. These numbers correspond to those on other Applied Biosystems DNA synthesizers which have 18 positions.
WARNING
Consider each chemical in the synthesizer potentially harmful. Do not inhale
vapors. Work in a well ventilated area. Always use eye protection and wear
acid-impermeable gloves and a lab coat. Do not leave any chemicals uncapped
If any chemical is ingested or comes in contact with the eyes, immediately
consult a physician. If there is any physical contact with a chemical, wash
immediately with water.
Please refer to the Material Safety Data Sheets (MSDS) in the Safety Data
Section for further information about the chemicals. The MSDS provide details
about physical characteristics, hazards, precautions, first aid, spill cleanup
and disposal procedures. In addition, refer to Section 2: How to Change the
Waste Bottle before waste disposal.
How to Store Reagents
Applied Biosystems chemicals must be stored properly to assure their stability. Improper storage
will result in reduced shelf life. All DNA synthesizer reagents, except the iodine solution, are guaranteed for one year from the date of manufacture. The iodine is guaranteed for six months from the
date of manufacture. Please follow the storage recommendations below.
Store at room temperature away from excessive heat and moisture:
• 1-methylimidazole, bottle 12
• Acetic Anhydride, bottle 11
• Trichloroacetic Acid, bottle 14
• Iodine, bottle 15
Store in a desiccator at room temperature not exceeding 25°C:
• All phosphoramidites, bottles 1 to 5
• All CPG columns
• Bulk CPG
• Tetrazole, bottle 9
• Anhydrous Acetonitrile
Section 2: Operation
2-19
Applied Biosystems
Store refrigerated at 4°C:
• Ammonium Hydroxide
Note
Cooler temperatures may cause crystal formation in the iodine or tetrazole solution
which could clog delivery lines. Gentle warming and agitation will dissolve these
crystals.
Bottles 1 to 5: Phosphoramidites
Reservoirs 1 through 4 contain phosphoramidites dissolved in anhydrous acetonitrile. They are used
to synthesize single species sequences and mixed sequence probes:
Contents
adenosine phosphoramidite (A)
guanosine phosphoramidite (G)
cytosine phosphoramidite (C)
thymidine phosphoramidite (T)
Reservoir
1
2
3
4
Position 5 is a spare phosphoramidite reservoir and can be used for synthesis with modified bases.
It is referred to as the “X port” or “X reservoir”. Many useful derivatives, such as deoxyinosine, are
available from Applied Biosystems and can be placed in this position. In addition, Aminolink 2™,
a linker for various substrates, can be placed in position 5.
IMPORTANT Since all phosphoramidite reservoirs are pressurized simultaneously with a single
valve, all five bottles must be attached to the instrument, even if some are empty, to
perform a synthesis.
Phosphoramidites are extremely sensitive to acid, oxygen and water. Once they are in solution and
the protective cap is removed, quickly put them on the instrument to prevent contamination.
IMPORTANT Use anhydrous acetonitrile with less than 90 ppm water to dissolve the
phosphoramidites. Do not use HPLC grade acetonitrile, its higher water content will
decrease coupling efficiency. Anhydrous acetonitrile is available from Applied
Biosystems, Part Number 400060.
Phosphoramidites are stable in powder form for one year and should be stored at room temperature
in a desiccator. Once they are dissolved, they should be used within approximately two weeks. After
this time, coupling efficiencies may decrease. If they cannot be used within this time, you can
freeze, store, thaw and then reuse the phosphoramidites. However, they may show a loss of activity.
For details, see How to Prepare Phosphoramidites and How to Store Dissolved Phosphoramidites
found earlier in this section.
2-20
Section 2: Operation
Applied Biosystems
Bottle 9: Tetrazole
Tetrazole (0.5 M) in anhydrous acetonitrile is used as the activator for the phosphoramidites. Once
the protective seal is removed, quickly put the tetrazole on the instrument to prevent atmospheric
water contamination.
IMPORTANT Tetrazole will form a precipitate at approximately 12°C. Do not put precipitated
tetrazole on the instrument. Examine each bottle before using it. If the tetrazole has
precipitated, warm it slightly until it is in solution.
In cold climates, the tetrazole may precipitate in the delivery lines. The resulting repairs are expensive, therefore, keep room temperatures above 18°C. Store tetrazole at room temperature.
Bottle 11: Acetic Anhydride
Acetic anhydride-lutidine-tetrahydrofuran (THF), (1:1:8) is one half of the capping reagent. Atmospheric water will reduce its efficiency. Upon opening the bottle, quickly place it on the instrument.
Use in a well ventilated area, avoid inhalation. Store at room temperature.
Bottle 12:1-methylimidazole (NMI)
1-methylimidazole (NMI) is the second half of the capping reagent. Within 24 hours of combining
NMI with the acetic anhydride reagent, the solution discolors and becomes viscous. To prevent this,
the two reagents are stored in separate reservoirs and are simultaneously added to the column to perform capping. Upon opening the bottle, quickly place it on the instrument. Use in a well ventilated
area and avoid inhalation. Store at room temperature.
Bottle 14: Trichloroacetic Acid
Trichloroacetic acid (TCA) - dichloromethane (3% wt/vol) is the detritylating reagent. Store at
room temperature and use with caution.
Bottle 15: Iodine
Iodine-water-pyridine-THF (0.1M:1:10:40) is the oxidizing reagent. It can be stored for up to 6
months from the date of manufacture. After this time, it may develop a tar (visible when the bottle
is turned over) and should not be used.
Note the expiration date on the bottle. Any iodine which develops the tar before the expiration date
will be replaced, without charge, by Applied Biosystems. Use in a well ventilated area and avoid
inhalation.
Section 2: Operation
2-21
Applied Biosystems
Bottle 18: Acetonitrile
HPLC grade acetonitrile is used to wash the support, valve blocks and delivery lines and to remove
nucleophiles from the support prior to the coupling step.
IMPORTANT Use HPLC or UV grade acetonitrile with a specification of less than 300 ppm of water.
Water contamination of even 1000 ppm will lead to a 1 to 2% decrease in coupling efficiency. If
unsure of the water content, either check the acetonitrile using the Karl Fischer method of titration
(requiring an automatic titration apparatus) or distill it over phosphorous pentoxide followed by redistillation over calcium hydride. Although Applied Biosystems supplies HPLC grade acetonitrile,
it may be more economical to purchase it from a local supplier, such as Burdick and Jackson, Part
Number 015.
Manual Deprotection and Cleavage Reagents
Ammonium Hydroxide
Concentrated ammonium hydroxide is used to deprotect the DNA and cleave it from the support.
Reagent grade ammonium hydroxide should be purchased in 500 mL bottles from a local source.
Store it tightly sealed and keep refrigerated. It should not be used for more than one month after
opening. Any loss of ammonia concentration decreases its effectiveness, therefore, only open it immediately before use. Use in a fume hood.
2-22
Section 2: Operation
Applied Biosystems
Reagents and Solvents
Bottle
Position
1
2
3
4
5
Reagent/Solvent
Part Number
Deoxyadenosine (dA-bz) phosphoramidite
β-cyanoethyl
β-cyanoethyl
β-cyanoethyl
(0.25g)
(0.5g)
(1.0g)
400600
400330
400326
Deoxyguanosine (dG-ib) phosphoramidite
β-cyanoethyl
β-cyanoethyl
β-cyanoethyl
(0.25g)
(0.5g)
(1.0g)
400601
400331
400327
Deoxycyctosine (dC-bz) phosphoramidite
β-cyanoethyl
β-cyanoethyl
β-cyanoethyl
(0.25g)
(0.5g)
(1.0g)
400603
400332
400328
Deoxythymidine (dT) phosphoramidite
β-cyanoethyl
β-cyanoethyl
β-cyanoethyl
(0.25g)
(0.5g)
(1.0g)
400602
400333
400329
Available for modified bases for example:
Deoxyinosine (β-cyanoethyl)
(0.25g)
400402
9
11
12
14
15
18
Tetrazole/acetonitrile (180mL)
Acetic anhydride/lutidine/THF
(180mL)
1-methylimidazole (180mL)
Trichloroacetic acid (450mL)
Iodine/water/pyridine/THF (200mL)
Acetonitrile 4-L*
Anhydrous Acetonitrile
(for dissolving phosphoramidites)*
Ammonium hydroxide
400606
400607
400785
400236
400753
400443
400060
400019
*It may be more economical to purchase ammonium hydroxide and HPLC grade acetonitrile from a local supplier.
About Deoxyinosine
Deoxyinosine base pairs with A, T and C, which makes it a suitable choice for synthesis where the
degree of degeneracy needs to be reduced. A 23-mer with five inosine substitutions used a as a
probe was found to bind to a complementary strand, and have similar dissociation temperature as a
Section 2: Operation
2-23
Applied Biosystems
17-mer. It is not well understood how deoxyinosine affects the stabilization of the DNA duplex,
however.
Martin, et al determined the stability of oligodeoxyribonucleotide duplexes with each of the four
normal bases using optical techniques for measuring melting temperatures.6 They observed large
neighboring-base effects upon the stability of the base pairs between inosine and the normal bases.
The results obtained by this group indicate that deoxyinosine reduces the specificity of hybridization probes.
Ohtsuka, from the faculty of Pharmaceutical Sciences and Institute for Molecular and Cellular Biology at Osaka, Japan, has found that synthetic oligonucleotides with deoxyinosine residues at ambiguous points are useful as hybridization probes.7
Takahashi, also from Osaka University, used a synthetic probe containing deoxyinosine to isolate
the cholecytokinin gene directly from a human genomic library.8
2-24
Section 2: Operation
Applied Biosystems
How to Change an Argon Tank
You can replace an empty argon tank before beginning a synthesis or when a synthesis has been
interrupted. Replace the empty cylinder as follows:
1. Shut off the cylinder at the tank and at the needle valve on the regulator.
2. Remove the line that connects the tank to the synthesizer by unscrewing the Swagelock
fitting.
3. Remove the regulator from the empty tank, clean the threads on the fittings, and install the
regulator on a full tank.
4. Turn the tank on and check for leaks at the connection of the tank to the regulator.
5. Open the needle valve.
Note
You may hear a hissing sound while the new argon supply is recharging the vacuum
assist.
WARNING
Be sure to anchor the cylinder securely and follow required safety practices
when handling gas cylinders.
How to Change the Waste Bottle
The synthesizer generates 1 to 2 liters of hazardous, halogenated, organic liquid waste per 100 base
additions. The waste is collected in a 4-liter polyethylene bottle which is placed on the floor or on
a nearby bench lower than the instrument. The bottle can be kept inside a protective carrier to contain accidental spillage. A one-gallon carrier is sufficient and can be purchased from VWR, Part
Number 56609-186; or Nalge, Part Number 6501-0010. When the bottle is full, it must be emptied
as follows:
1. Unscrew the cap assembly and immediately recap the bottle to prevent the release of vapors.
Save and reuse old bottle caps.
2. Discard the waste. Place the liquid in a sealed container labeled “FLAMMABLE,” “POISON
B N.O.S.” or absorb in vermiculite, dry sand or earth. Dispose of the waste following
applicable government regulations.
WARNING
Section 2: Operation
Synthesizer waste must be disposed of properly and carefully.
When handling the waste for disposal, wear gloves and eye
protection, and avoid inhalation and skin contact. Refer to the
Waste Profile and MSDS in the User’s Safety Information Section
for details.
2-25
Applied Biosystems
3. After disposal, securely screw the cap assembly supplied by Applied Biosystems on an empty
waste bottle.
IMPORTANT
The waste bottle is the low pressure side of the delivery system and
must always be kept vented to atmosphere. Be sure the vent line is
properly routed to a fume hood. If the vent line is blocked, back pressure will be generated which will decrease the deliveries of reagents
and solvents.
How to Perform the Flow Test Procedure
Flow test is an automated procedure used to measure the flow rates through all essential delivery
lines. It can be used for routine maintenance, troubleshooting and recalibration of the instrument if
flow related problems are suspected. For example, if oligonucleotide quality is low, the flow test
will help determine if instrument performance is a factor. If the synthesizer has flow related difficulties, be sure to do the test prior to calling the Applied Biosystems Technical Support Department.
The flow test has four parts and takes about one hour to perform. The first part washes and primes
the delivery lines with acetonitrile. The second measures the flow of acetonitrile from each bottle
position to the lower column Luer fitting. The third measures the flow from bottle 14 to the trityl
collection line. In the final part, all the lines are flushed dry with argon enabling reattachment of the
reagents.
The flow test is not found in the procedure editor and cannot be edited or printed. A copy of the
procedure can be found in Appendix A.
How to Do the Flow Test Procedure
From the Main Menu, select SELF TEST and then press MORE until the display shows:
Select a test . . .
Version 1.00
flowtst
more
main
Press FLOWTST and the screen will read:
Remove all bottles except #18 before you
begin flowtst
2-26
begin
more
main
Section 2: Operation
Applied Biosystems
Follow the instructions and remove all reagents except bottle 18, acetonitrile. Do not use the change
bottles procedure. Any reagents that will be reused should be purged with argon and capped tightly.
Part 1
Press BEGIN, the procedure will start and the screen will show step one:
Step:1
hold
WAIT
jump
5 of 5 sec
intrpt
more
main
The first series of steps, 1 through 14, sequentially rinses the lines for each bottle position. Use a
beaker to collect the acetonitrile rinse starting with position 15 and working backwards. Each rinse
takes 20 seconds. Watch the time on the screen to know when to move the beaker to the next position. Step 12 delivers acetonitrile to the column. Collect this rinse at the lower Luer fitting (remove
the column if necessary).
Inspect the type of flow coming from each delivery line. Acetonitrile should flow from positions
15, 14, 12 and 11 and should drip steadily from positions 1 through 5 and 9. If the flows are not as
described, retest each faulty position by using the JUMP key. Refer to Section 3: Main Menu Option: Monitor Synthesis for details on how to use JUMP.
At step 14, the procedure is interrupted and the screen reads:
Step:14
resume
INTERRUPT
jump
abort
1 of 1 sec
more
main
Part 2
This pause is used to prepare for steps 15 through 43, part two of the procedure. During this part the
flow rate from each reservoir is measured for 2 minutes at the lower column Luer fitting.
Before beginning, attach a clean bottle filled with HPLC grade acetonitrile to each bottle position.
A set of clean, empty bottles is shipped with the instrument. Next, remove the column (if necessary)
and place a 10-mL graduated cylinder under the lower column Luer fitting. When ready, press RESUME and the procedure will continue.
At step 17, acetonitrile will flow from Reservoir 1 (A-phosphoramidite) to the lower Luer for exactly 2 minutes. Carefully collect the flow. A 20-second WAIT step immediately follows. Use this
time to read and record the measurement. In addition, empty the cylinder and replace it under the
lower Luer for the next reading. If 20 seconds is too short, simply press HOLD during the WAIT
step. Then, when you are ready to continue, select RESUME. Repeat this procedure until all mea-
Section 2: Operation
2-27
Applied Biosystems
surements are taken. Please note that step 40, #18 to waste, is used during manufacturing and does
not require measurement.
Calculate the flow rate in milliliters per minute. Remember, you are collecting for 2 minutes so you
must divide the mL amount by 2 to get mL/min. The correct flow rates are listed below. If they are
not within specification, the argon pressure regulator must be adjusted and the flows remeasured.
BOTTLE POSITION
1 to 5, 9
11, 12
14
15
18
FLOW RATE
(mL/min)
1.90 to 2.10
2.80 to 3.00
2.70 to 3.10
2.80 to 3.00
2.80 to 3.20
Note that since the phosphoramidite reservoirs are pressurized simultaneously, their flow rates
should be quite similar. If one flow rate is very different, the delivery line for that phosphoramidite
may be clogged. If you suspect this, do not change the pressure setting. Be sure to measure all flow
rates and then call the Applied Biosystems Technical Support Department.
To adjust the pressure regulator, first remove the left side panel of the instrument. The regulator
knob is located to the left of the top of the ballast assembly. The ballast assembly is shown as item
14 in Figure D-2, in Appendix D. To access the regulator gauge, remove the back panel of the instrument. The gauge is shown as item 15a in Figure D-3, in Appendix D. The regulator is usually
set at approximately 4.0 psi.
If the flow rate is too slow, increase the regulator setting by turning the knob clockwise approximately 1/8th of a turn. Using JUMP, perform the delivery step for the faulty position for at least 90
seconds to equilibrate the pressure. For example, if the A-phosphoramidite flow rate is too slow,
increase the pressure as just described and jump to step 17, A to column, for equilibration. Next,
using JUMP again, repeat the delivery step (i.e. step 17, A to column) and remeasure the flow rate
for 2 minutes.
Similarly, if the flow is too fast, decrease the regulator setting by turning the knob counterclockwise
about 1/8th of a turn. Next, remove the bottle to relieve the excess pressure and then replace it. As
above, equilibrate the bottle pressure for 90 seconds and remeasure the flow rate for two minutes.
Once the pressure has been changed, remeasure each position to be sure all flow rates are within
specification.
If all flow rates cannot be adjusted to specification or if you do not feel comfortable adjusting flow
rates, please call the Technical Support Department. Do not use the synthesizer until all flow rates
are correct.
2-28
Section 2: Operation
Applied Biosystems
Part 3
At step 43, the procedure is interrupted again. Use this time to prepare for part three, steps 44 to 46.
Connect the upper and lower column Luer fittings with the clear plastic shipping tube provided with
the instrument. Place the trityl collection line inside a beaker. Place a 10-mL graduated cylinder
next to the beaker.
When you are ready, select RESUME. This step is timed for 150 seconds. Use the first 30 seconds
to prime the line. Then watching the time on the display, place the trityl collection line inside the
10-mL graduated cylinder and collect the flow for 120 seconds. Calculate the flow rate in milliliters
per minute. The flow rate should be 2.30 to 2.60 mL/min. If the flow rate is not correct, there may
be a problem with the post-column valve block. If you suspect this, call the Technical Support Department for help.
Part 4
The procedure is interrupted again at step 46. In part 4, steps 47 to 59, the delivery lines and valve
blocks are flushed dry with argon. Press RESUME and the residual acetonitrile will be collected in
each bottle starting at position 15 and going backwards.
The test is now complete. Reagents can be reattached using the Change Bottles Menu. As each flow
test bottle is removed, wipe the exposed delivery line with a lint-free tissue. Once all chemicals are
attached, synthesis can begin.
Section 2: Operation
2-29
Applied Biosystems
Aminolink 2™ Reagent
Introduction
Aminolink 2 is a DNA synthesis reagent which affords a convenient method to introduce an aliphatic primary amine at the 5' end of oligonucleotides. This amine can react to form oligonucleotide
conjugates with a variety of substrates such as biotin9,10, fluorescent dyes11, EDTA12, or alkaline
phosphatase.13 Applications include:
• Non radio-labeled hybridization probes9,10,13,14
• Sequence specific cleavage of single stranded DNA12
• Automated DNA sequencing - affinity chromatography
Aminolink 2 offers many advantages. Because it is a phosphoramidite, it reacts like conventional
nucleoside phosphoramidites. The compound is dissolved in anhydrous acetonitrile and placed on
the extra phosphoramidite port (bottle position 5, X). It is activated with tetrazole and couples with
the same high efficiency found in standard oligonucleotide synthesis. In addition, when using Aminolink 2 you do not need to modify the synthesis cycle. Other features include:
• Solution stability is comparable to nucleoside phosphoramidites (at least 2 weeks).
• No special cycles or protocols are required.
• Labeling at the 5'-terminal amine does not interfere with hybridization.
• Aminolinked oligonucleotides can be easily labeled with a variety of commercially available
reagents such as fluorescein isothiocyanate and biotin NHS ester.
Chemical Description
Aminolink 2 (structure shown in Figure 2-2) generates an amino hexyl phosphate linker with a
methoxy phosphate protecting group. Prior to deprotection, the phosphite triester is oxidized to the
phosphate triester using standard oxidizing reagents. The amine group is protected by a trifluoroacetyl group, which is removed during standard ammonia deprotection procedures.
2-30
Section 2: Operation
Applied Biosystems
Figure 2-2.
Aminolink 2 — Oligonucleotide
Section 2: Operation
2-31
Applied Biosystems
How to Use Aminolink 2
Aminolink 2 (Part Number 400808) is supplied in 250 mg quantities. Store the unopened linker desiccated away from heat and moisture.
1. Dilute Aminolink 2 with 3.3 mL of anhydrous acetonitrile following the instructions in How
to Prepare Phosphoramidites. (Once diluted and placed on the instrument, the compound is
stable for at least 2 weeks.)
2. Place Aminolink 2 on bottle position 5.
3. Using the DNA Editor, enter X (the Aminolink bottle position), as the first base, 5' end of the
sequence.
4. Configure the synthesis “Trityl ON” since detritylation is not necessary after Aminolink 2
addition.
5. Deprotect and cleave the Aminolink-oligonucleotide following the instructions in the Manual
Deprotection and Cleavage Procedure. Although Aminolink has a methyl phosphate
protecting group, thiophenol deprotection is not necessary. Ammonia treatment will
demethylate the single methyl phosphotriester group of the Aminolink-oligonucleotide
without adversely affecting product purity.
Analysis and Purification of Aminolink™-Oligonucleotides
Aminolink-oligonucleotides can be analyzed and purified by the same methods used for conventional oligonucleotides. Since the 5'-hydroxyl, usually available for phosphorylation by kinase and
ATP, is blocked by the linker, an alternate 3'-end labeling procedure must be used to radiolabel
Aminolink-oligonucleotides. To effect a 3'-end label, the enzyme Terminal Deoxynucleotidyl
Transferase (TDT), is used. TDT adds a single nucleotide to the 3'-hydroxyl. Because the enzyme
would continue propagation beyond the first nucleotide addition, a dideoxynucleotide analogue of
[alpha-32P]-dATP is used. The analogue, [alpha-32P]- Cordycepin-5'- triphosphate, does not have a
3'-hydroxyl and therefore further addition is prevented. Below is a protocol for the labeling procedure.
3'-End-Labeling
1. Prepare a stock solution of 10X 3'-end-labeling buffer:
1.4 M Sodium Cacodylate*
10 mM Cobaltous Chloride
1.0 mM Dithiothreitol
pH 7.5 with HCl
*This chemical is carcinogenic, handle with care.
2-32
Section 2: Operation
Applied Biosystems
2. Aliquot 2 pmol of oligonucleotide into an Eppendorf-type tube (1.5 mL), dry in a Speed-Vac
and ethanol rinse with 10 µL of 95% Ethanol, dry.
3. Add the following to the tube, while on ice:
1 µL of 10X 3'end-labeling buffer
1 µL of [alpha-32P]-Cordycepin-5'Triphosphate
(New England Nuclear #NEN-114, specific activity greater than 5000Ci/mmol)
7 µL of deionized water
1 µL of TDT (ENZO Biochemicals, 5 units per microliter)
4. Spin the tube very briefly in a microcentrifuge to bring contents of tube to the bottom.
Incubate sample at 37°C for one hour.
5. Add 25 µL of 1.0 mM EDTA in deionized formamide loading buffer to the sample.
Bromophenol Blue and Xylene Cyanol can be added as marker-dyes to the formamide prior
to addition to the sample. The sample can be loaded onto an analytical gel in the same manner
as described in Applied Biosystems DNA Synthesis User Bulletin No. 13 (Revised), Analysis
and Purification of Synthetic Oligonucleotides. The linker retards the migration of the
oligonucleotide by approximately one-half of a base in the gel.
There is occasionally a radioactive contaminant which is a by product of the labeling procedure.
This contaminant is most likely a type of enzyme-Cordycepin complex which co-electrophoreses
with the sample and may appear on the autoradiogram as a fuzzy band in the region of a 16-mer. It
is recommended that a blank sample containing the constituents of the reaction but without DNA be
loaded on the gel so that the contaminant can be compared with the DNA sample. Note that since
phosphorylation of the 5-hydroxyl by T4 kinase should be blocked by the presence of the linker,
this procedure can be used as a qualitative analysis of the efficiency of Aminolink 2 addition.
Anion-exchange HPLC, described in User Bulletin No. 13 (Revised) can be used for Aminolinkoligonucleotide analysis or purification. The inherent problem with using reverse phase: HPLC for
purification is that the oligonucleotide and the Aminolink oligonucleotide are not well resolved. Gel
purification seems to give better results. Once a dye is attached, reverse phase HPLC may be used
to purify the derivatized oligonucleotides.
Section 2: Operation
2-33
Applied Biosystems
Applications
General Protocol for Tag/Labeling Substrate
Described below is a generic protocol from which to extrapolate individual application protocols.
Note that most commercially available biotinylation reagents come with usage protocols.
1. Prepare a solution of Aminolink-oligonucleotide at a concentration of 1mM or greater in a
50mM-200mM-NaHC03/Na2CO3 Buffer, pH 9.
2. Add a 50-molar excess of the solid tag/labeling substrate (NHS-Biotin, Dye, etc.) and vortex
to dissolve. If the tag or DMSO is insoluble in aqueous solutions, it can be dissolved in
dimethylformamide before addition to the Aminolink-oligonucleotide. The final
concentration of organic solvent in the reaction mixture should not exceed 30%.
3. After several hours reaction time at room temperature, the mixture can be purified by gelexclusion in a G-50 or G-25 Sephadex™ column to remove the excess tag. Use 100mM
TEAA as the eluant.
Note
2-34
Reactions with fluorescent dyes should be done in the dark.
Section 2: Operation
Applied Biosystems
How to Use OPC (Oligonucleotide Purification Cartridge)
Introduction
OPC (Part No. 400771) is a rapid purification cartridge used specifically for synthetic DNA. It provides the level of purity required for the common applications of synthetic DNA. Using OPC eliminates the need to purify by time-consuming and labor intensive polyacrylamidge gel
electrophoresis (PAGE) or HPLC. If you currently only desalt your synthetic oligonucleotides, you
can now use OPC to desalt and purify in less time than it takes to desalt alone.
OPC is fast, easy to use and delivers consistent results. The time required to go from the deprotected, crude oligonucleotide to its use in an experiment is greatly reduced. Complete purification requires only 15 minutes.
After completing deprotection and cleavage, an aliquot of the crude oligonucleotide, still in ammonia is diluted with water and then loaded directly onto the OPC cartridge. This eliminates the need
to evaporate the sample prior to purifying. The remaining portion of the crude synthesis can then be
stored in ammonia either refrigerated or at -20°C without harming the DNA. In fact, this is preferable to storing it as an evaporated sample or dissolved in water. The ammonia protects the sample
from acidic by products present in the crude.
Detritylation is also done on the cartridge, eliminating the need for post-purification work-up. The
purified oligonucleotide is then eluted as a trityl-off species in a volatile buffer. Quantification can
be easily accomplished and only the volume containing the amount required for the experiment
need be evaporated. The reminder can be stored at -20°C for later use. Once dry, the pure oligonucleotide can be redissolved in an appropriated buffer and used in your experiment. The product obtained from OPC is highly-purity DNA that can be used with complete confidence.
The OPC purification protocol appears at the end of this section. It successfully purifies sequences
up to 70 bases. Note that for sequences 40-70 bases long, you must perform an additional step, number 9a. This step adds several washes with dilute ammonia after detritylation with 2% trifluoroacetic
acid. The extra basic wash removes preferentially any shorter sequences that had trityl protecting
groups attached and thus co-purified with the desired product.
The purification of longer oligomers (>70-mers) using OPC can be more difficult. But, a protocol
has been developed by Thomas Horn and Mickey Urdea of Chiron Co.15 in which you pre-treat the
oligonucleotides with lysine and then perform the OPC purification procedure. (See the Protocol
for Lysine Pre-treatment of Longmers (>70 bases).) The usefulness of the lysine pre-treatment is
dependent on the amount of depurination in the oligonucleotide. The greater the depurination, the
more useful the procedure might be. Depurination is sequence and reagent dependent. The higher
the purine content of a sequence, the greater the chances are of depurination occurring. The higher
Section 2: Operation
2-35
Applied Biosystems
the reagent purity, the smaller the chances are of depurination occurring. Because of Applied Biosystems high purity reagents, depurination is usually not detectable.
More about lysine treatment of longmers (>70 bases) before OPC purification:
For long oligomers (even with very high synthesis efficiency) the desired, full length product is a
minor component compared to the sum of the failure sequences. Certain side reactions also become
more prevalent due to increased exposure to the synthesis reagents. In particular, the acids used for
detritylation (trichloroacetic or dichloroacetic acid) can promote cleavage of the purine nucleobases
(A and G) from the ribose rings. During synthesis of a long-mer, the cumulative time that the bases
near the 3' end are subjected to acid can be more than an hour. During this time apurinic sites can
be created. These apurinic sites in the oligonucleotide undergo internucleotide cleavage during ammonia deprotection at 55°C. When the synthesis is conducted Trityl On, in preparation for OPC purification, some of the cleavage products will bear a 5'-trityl group (see Figure 2-3). Apurinic
cleavage can generate trityl bearing species that are less than full length. Reverse phase HPLC or
OPC purification methods, which depend on trityl selectivity, are then less efficient in purifying the
full-length oligo.
3'
CPG
CPG
AGTCAGTGCGCTTAGCCATAAG
3' AGTC*GTGCGCTTAGCCATAAG
-2
5'
5'
CPG + 3' AGTC-OPO + GTGCGCTTAGCCATAAG
3
DMT (full-length oligo)
DMT (oligo with apurinic site)
5'
DMT (ammonia cleavage
products)
Figure 2-3.
Thomas Horn and Mickey Urdea of Chiron Co. published a procedure for extending the utility of
OPC to the purification of long oligonucleotides15. They found that apurinic sites undergo internucleotide cleavage with 1 molar lysine, without cleavage of the oligonucleotide from the support.
(See the Protocol for Lysine Pre-treatment of Longmers (>70 bases).) The DMT bearing cleavage
fragment is washed away, leaving a non-DMT bearing fragment bound to the support. Following
ammonia cleavage and deprotection, they used the OPC purification protocol. This was effective in
purifying oligomers up to 118 bases in length.
Although depurination is a valid concern during DNA synthesis of long oligonucleotides, the degree of depurination encountered during synthesis is highly sequence and reagent dependent. Since
2-36
Section 2: Operation
Applied Biosystems
the 3' bases of an oligo (the initial couplings) have the greatest reagent exposure, varied purine content in this area will generate varied potential for depurination. Another factor in depurination is the
purity of the acid used for detritylation. Contaminants, such as water or HCl, in the trichloroacetic
or dichloroacetic acid will greatly promote depurination. Applied Biosystems supplies high purity
trichloroacetic and dichloroacetic acid reagents. In a study involving the synthesis of a 72-mer, the
use of Applied Biosystems reagents did not promote detectable depurination. Analysis of the 72mer synthesis, OPC purified with and without prior lysine treatment, showed no detectable purity
difference. These results may not apply to all synthesis sequences and lengths, but the study demonstrates Applied Biosystems commitment to synthesis reagent and product quality.
Protocol for Lysine Pre-treatment of Longmers (>70 bases)
1. Synthesize the oligonucleotide Trityl ON.
2. Dry the column by reverse flushing (function 2) for 60 seconds with argon or allow the
column to air dry.
3. Remove the phosphate protecting groups: Connect a syringe (with plunger) to one end of the
column. In a fume hood, fill another syringe with the following solution, attach it to the
synthesis column and fill the column with the solution.
For cyanoethyl phosphoramidites: add 1 ml of t-butylamine:pyridine (1:9 v/v) and treat at
room temperature for 1 hour (t-butylamine; Aldrich B8,920-5 and pyridine;
Aldrich 27,040-7).
Periodically circulate the solution by alternately depressing the syringe plungers.
Note
Gently depress the syringe plungers; otherwise you risk breaking the filters that
hold the CPG.
4. Remove the solution and wash 5 times with acetonitrile.
5. Allow the column to air dry or blow the column dry with argon.
6. Prepare a 1M lysine solution pH = 9 (adjust pH with NaOH if necessary)
(L-lysinemonohydrochloride; Sigma # L5626, Aldrich # L460-5).
7. Place 2 ml of the lysine solution into a 16 X 100 mm test tube. Connect a single syringe to one
side of the synthesis column and a male-to-male luer fitting (AB p/n 110127) the other. Insert
the column-syringe-luer apparatus into the test tube and draw the lysine solution into the
column. Incubate at 55°C for 90 minutes by inserting the test tube and contents into a heating
block. The lysine solution should be periodically circulated (every 30 minutes).
Note
The luer fitting of the syringe must be located in the center of the syringe barrel
to allow easy insertion into the test tube.
8. After incubation in the lysine solution, wash the column 5 times with water.
9. Complete the Manual Deprotection and Cleavage Procedure described earlier in this section.
Then follow the OPC purification protocol.
Section 2: Operation
2-37
Applied Biosystems
OPC Purification Protocol
Solutions Needed
• HPLC grade acetonitrile, 5 mL
• 2.0 M triethylamine acetate (Part No. 400613), 5 mL
• Deionized water, 1 mL
• Dilute ammonium hydroxide, 15 mL (1:10 dilution of conc.
ammonium hydroxide in deionized water)
• 2% trifluoroacetic acid in deionized water, 10 mL (Neat TFA Part
No. 400137)
• 20% v/v acetonitrile in deionized water, 1 mL
1. After completion of a Trityl On synthesis, cleave the
oligonucleotide from the support and deprotect following the
Manual Deprotection and Cleavage Procedure found earlier in this
section.
2. Connect an all-polypropylene syringe (Aldrich Zl 1686-6) to one
end of the OPC column and a male-to-male Luer tip to the other
end. See Figure 2-4. Make sure all fittings are snug.
3. Flush the cartridge with 5 mL HPLC grade acetonitrile, followed
by 5 mL 2.0 M triethylamine acetate.
Note
Keep the flow rate at 1 to 2 drops per second for all
reagent additions.
4. Dilute the ammonium hydroxide solution containing the cleaved,
deprotected, “trityl-on” crude oligonucleotide with one-third
volume of deionized water.
Figure 2-4.
5. Load 20 - 40 OD’s of the above solution into the syringe and then
gently push it through the cartridge, saving the eluted fraction.
Reload this fraction, and again push it through the cartridge.This
will load 1 to 5 OD units of the trityl oligonucleotide (depending on
length, sequence, and synthesis quality) onto the cartridge. Save
this final eluted fraction in ammonia at -20°C, it can be put through
another cartridge until exhausted of trityl oligonucleotide.
6. Flush cartridge with 3 x 5 mL dilute ammonium hydroxide.
7. Flush cartridge with 2 x 5 mL deionized water.
2-38
Section 2: Operation
Applied Biosystems
8. Detritylate the OPC-bound oligonucleotide with 5 mL of the 2% trifluoroacetic acid solution.
Gently push ~1mL through the cartridge, incubate for 5 minutes, then flush the remaining
TFA solution through the cartridge.
9. Flush cartridge with 2 x 5 mL deionized water.
10.(For sequences ≥40 bases, add this step) Flush cartridge with 1 x 5 mL dilute ammonium
hydroxide, then 2 x 5 mL deionized water.
11. Elute the purified, detritylated oligonucleotide by flushing the cartridge with 1 mL of the 20%
acetonitrile solution.
12.Evaporate to dryness an aliquot of the eluate (step 10) and dissolve in water to determine the
OD units at A-260.
13.Store any unused OPC-purified oligonucleotide as a dry solid at -20°C.
Section 2: Operation
2-39
Applied Biosystems
References
1. --------------, Evaluation and Purification of Synthetic Oligonucleotides, Applied Biosystems
User Bulletin 13 (Revised) (1987).
2. Gait, M.J., and Sheppard, R.C., Nucleic Acids Research 4, 4391 (1977).
3. Tanaka, T. and Letsinger, R.L., Nucleic Acids Research 10, 3249 (1982).
4. Schott, M.E., A simple manual method for oligonucleotide synthesis, American
Biotechnology Laboratory 3, 20-23 (1985).
5. McBride, L.J., Eadie, J.S., Efcavitch, J.W., and Andrus, W.A., Base modification and the
phosphoramidite approach, Proceedings of the 7th International Round Table on Nucleosides,
Nucleotides and Their Biological Applications, (1986).
6. Martin, F.M., Castro, N.M., Aboula-ela, F., Tinoco, I., Base Pairing Involving Deoxyinosine:
Implications for Probe Design, Nucleic Acids Research 13, 8927-8938, (1985)
7. Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y., and Matsubara, K., An Alternate
approach to Deoxyoligonucleotides Hybridization Probes by Insertion of Deoxyinosine at
Ambiguous Codon Positions, J. Biol. Chem. 260, (5), 2605-2608, (1985)
8. Takahashi, Y., Kato, K., Hayashizaki, Y., Wakabayashi, T., Ohtsuka, E., Matsuka, S., Ikehara,
M., and Matsubura, K., Molecular Cloning of Human Cholecytokinin Gene by Use of a
Synthetic Probe Containing Deoxyinosine, PNAS 82, 1931-1935, (1985)
9. B.C.F. Chu and L.E. Orgel, DNA 4
327-331 (1985).
10.A. Chollet and E.H. Kawashima, Nucleic Acids Research 13
11. L.M. Smith et al., Nature 321
1529-1541 (1985).
647-679 (1986).
12.G.B. Dreyer and P.B. Dervan. Proc. Natl. Acad. Sci. USA 82
13.E. Jablonski, et al., Nucleic Acids Research 14
14.B.A. Connolly, Nucleic Acids Research 15
968-972 (1985).
6115-6128 (1986).
3131-3137 (1987).
15. Horn, T. and Urdea, M. S., Nuc. Acids Res. 16 (24), 11559-11574 (1988).
2-40
Section 2: Operation
Section 3:
Software Menu Descriptions
This section describes each menu in detail. The most frequently used menus are the DNA Editor,
Start Synthesis, Monitor Synthesis, and Change Bottles. Be sure you carefully read about these
menus. You can use this section while viewing the synthesizer.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2
Software Abbreviations and Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
The Main Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5
Summary of Main Menu Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6
Main menu options in detail:
Main Menu Option: DNA Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-7
Main Menu Option: Start Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-13
About the Ending Method: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-14
Main Menu Option: Monitor Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-16
Instrument Status: Synthesizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-17
The Holding Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-19
The Jump Step Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-22
The Interrupt Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-24
Main Menu Option: Change Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-29
About the Alarm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-31
Bottle Usage Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-33
Main Menu Option: Cycle Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-34
The Base Specifier Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-37
Main Menu Option: Manual Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-40
Main Menu Option: Fract Pulse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-42
Main Menu Option: Procedure Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-42
Main Menu Option: FXN Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-44
Main Menu Option: Power Fail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-47
Main Menu Option: Self Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-48
Flow Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-50
Main Menu Option: Set Clock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-51
Main Menu Option: Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-52
Applied Biosystems
Introduction
The Model 391 software is “menu driven”. Menus and pages of menus are shown on a 2-line liquid
crystal display (LCD). The menus present various options and necessary information about the synthesis or status of the instrument. In response, you select an option and give instructions by pressing
the appropriate key on the keyboard.
Figure 3-1.
The Keyboard
The keyboard has several labeled keys (0-9; A, G, C, T, X; delete; left and right arrows) and 5 “soft
keys” (directly below the display). The labels and actions of the soft keys change depending on the
information displayed directly above them.
Pressing one of the gray keys labeled 0 through 9, enters a numeric value at the
cursor position.
0-9
A
C
G
T
Pressing the appropriate gray key, labeled A, G, C, T or X, enters a base in a
DNA sequence. These keys operate when viewing the DNA Editor Menu.
X
--->
<---
To make entries or deletions, the cursor must be in the correct position and often must be moved. Pressing the right (→) or left (←) arrow key moves the cursor one position in the arrow’s direction.
Continuously pressing an arrow repeats the action. In menus that have multiple
entry areas, arrows also move the cursor from one entry area to another.
3-2
Section 3: Software Menu Descriptions
Applied Biosystems
Pressing the delete key erases an entry at the cursor position. When the cursor
is not under an entry, the deletion is made to the left of the cursor. Continuously
pressing delete will repeatedly erase entries.
DELETE
In addition to using the delete key to erase numerical entries, mistakes can be corrected by moving
the cursor to the incorrect number and typing over it.
The MAIN and MENU Soft Keys
The MAIN and MENU keys appear in the far right positions of most displays.
MAIN
Pressing the MAIN always returns to Page 1 of the Main Menu.
MENU
Pressing MENU returns to the display which was previously shown. MENU
can be selected repeatedly until the MAIN key appears.
Section 3: Software Menu Descriptions
3-3
Applied Biosystems
Software Abbreviations and Symbols
3-4
#
number
1µM
one-micromole β-cyanoethyl synthesis cycle
.2µM
two-tenth micromole β-cyanoethyl synthesis cycle
10µM
ten-micromole β-cyanoethyl synthesis cycle
#1 A-PHOS
bottle change for reservoir 1 containing adenosine
phosphoramidite (as seen in Change Bottles)
#1 BOTTLE CHANGE
bottle change procedure for reservoir 1 containing adenosine
phosphoramidite (as seen in the Procedure Editor Menu)
C1
Cycle-1
CLR
clear
Fract pulse
fraction collector pulse time
Fxn
function
Imediate
immediate
Intrpt
interrupt
Low
low consumption, two-tenth micromole β-cyanoethyl
synthesis cycle
Nxt cyc
next cycle
Nxt DNA
next DNA strand
#PP PHOS PURGE
phosphoramidite purge procedure
Prev
previous
Proc
procedure
RAM
random access memory
ROM
read only memory
#SD SHUT DOWN
shut down procedure
Std
standard
Synth
synthesis
Section 3: Software Menu Descriptions
Applied Biosystems
The Main Menu
Once the instrument is connected to a power source and the power is turned on, the following display appears:
Applied Biosystems 391 DNA Synthesizer
PCR-MATE EP
Ver. 1.00
start
Select START and the screen will display Page 1 of the Main Menu which presents five options:
DNA
start
change
cycle
more
editor
synth
bottles
editor
menu
Selecting MORE displays Page 2 of the Main Menu and shows additional options:
manual
start
proc
fxn
more
control
pulse
editor
editor
menu
Selecting MORE again displays Page 3 of the Main Menu and shows additional options:
power
self
set
shut
more
fail
test
clock
down
menu
Selecting MORE again returns to Page 1 of the Main Menu.
All other menus or menu pages are accessed by selecting the appropriate key from Page 1, 2 or 3 of
the Main Menu. To move from one menu (e.g., DNA EDITOR) to another (e.g., START SYNTH)
requires returning to the Main Menu by pressing the MAIN key. Note that sometimes, MENU is
shown instead of MAIN. Pressing MENU will return to the previous menu. Continue to press
MENU until the MAIN key is displayed.
Section 3: Software Menu Descriptions
3-5
Applied Biosystems
Summary of Main Menu Options
DNA
EDITOR
Creates, edits and prints up to four DNA sequences.
START
SYNTH
Initiates the steps to begin a synthesis. From this menu, you specify the cycle,
ending method and DNA strand you want to synthesize.
MONITOR
SYNTH
Displays the status of the instrument during a synthesis. Use it to identify
which base is being added and which step and function are being performed.
Use Monitor Synth to interrupt or abort a synthesis and to manipulate the synthesis cycle by using the HOLD and JUMP keys. In addition, during synthesis
the ending method can be changed from page 4 of this menu.
Note
When the instrument is ready to begin a synthesis, START SYNTH is displayed on
Page 1 of the Main Menu. However, during a synthesis, MONITOR SYNTH is shown
in that position.
CHANGE
BOTTLES
CYCLE
EDITOR
MANUAL
CONTROL
3-6
Provides a procedure for removing empty reservoirs and replacing them with
bottles of fresh reagents. It also shows bottle usage and provides an alarm to
alert you that reagent levels are low.
Creates, edits and prints synthesis cycles.
Used to manually activate functions and to open and close individual valves.
FRACT
PULSE
Changes the pulse time which advances the fraction collector.
PROC
EDITOR
Used to create, edit and print the bottle change, phosphoramidite purge and
shut down procedures.
Section 3: Software Menu Descriptions
Applied Biosystems
FXN EDITOR
Used to create and edit user functions and to print the standard, user and test
functions.
POWER FAIL
Used to program the instrument to automatically restart synthesis following a
power outage or to interrupt the synthesis until you restart or abort it.
SELF TEST
Verifies the correct operation of the electronic components of the instrument.
Access the Flow Test procedure.
SET CLOCK
Used to enter the current time and date.
SHUT DOWN
Prepares the instrument for long term storage by washing and drying all chemical pathways.
Main Menu Option: DNA Editor
The DNA EDITOR is used to create, edit and print up to four DNA sequences. The sequences or
strands are named DNA-1, DNA-2, DNA-3 and DNA-4. When DNA EDITOR is selected, the
screen displays one of the DNA strands and shows the number of bases in that strand. For example,
the following menu shows DNA-1 which currently has zero bases:
Select action for
edit
copy
print
DNA-1
0-mer
next
main
Follow the prompt and select an action for DNA-1:
EDIT
Select to create, edit or delete DNA strand designated in the menu.
COPY
Select to copy the designated DNA strand into any other DNA strand.
PRINT
When printer is connected to the instrument, select this option to print the designated DNA strand.
NEXT
Select to access the next DNA strand for subsequent editing, copying, and/or
printing.
Section 3: Software Menu Descriptions
3-7
Applied Biosystems
The Edit Key
When EDIT is selected, the display reads:
1 -5'>_
(
<3' 0
space
)
erase
menu
The 5' and 3' positions are clearly marked and the cursor is to the right of 5'>. The number at the top
left corner of the screen signifies which DNA strand is being shown (i.e. 1 = DNA-1). The number
at the top right comer of the screen indicates the base position number. It equals zero when there
are no entries and increases by one as each base is entered. Also, it displays the base number corresponding to the cursor position.
For example, when the cursor is under C in the sequence 5'AGCT 3', C is shown as base number 3:
5'>AGCT
<3' 3
IMPORTANT Following standard convention, The DNA sequence is entered 5' to 3'. The DNA
Editor Menu displays the 5' terminus as Base 1 and subsequent bases are numbered
5' to 3'. However, the DNA strand is actually synthesized 3' to 5'.
To enter a sequence, begin by pressing the gray key (labeled A, G, C, T or X) which corresponds
to the 5' terminus. The base will appear on the screen, the cursor will move one position to the right
and the base position number will increase to one. The display will read:
1 -5'>A_
(
<3' 1
space
)
erase
menu
Continue to enter the sequence (5' to 3') until it is the specified length. When finished, check it carefully to be sure it is correct. If possible, print the sequence to verify it.
(
)
Use parentheses to insert multiple bases in one position for synthesizing mixed
sequence probes.
This is done by selecting the left “open” parenthesis, “( ”, typing any combination of bases A, G,
C, T or X; and then selecting the right “close” parenthesis, “)”. For example, to insert AGCT in the
third position of the sequence 5'AA 3':
3-8
Section 3: Software Menu Descriptions
Applied Biosystems
select
(
5'>AA_
<3';
; 5'>AA(_
<3';
press A, G, C, T ; 5'>AA(AGCT_
select
)
; 5'>AA(AGCT)_
<3';
<3'; continue entering the sequence
Multiple bases are added to the support-bound nucleotide by simultaneous delivery of the specified
phosphoramidites. They can be placed in any position except the 3' terminus.
SPACE
Inserts a space to the left of the cursor position. For example, to insert a space
following C in the sequence 5'AGC 3':
5'>AGC_ <3'
press space
5'>AGC
_ <3'
To insert a space between C and T in the sequence 5'AGCTAG 3':
5'>AGCTAG <3' press space
5'>AGC TAG
<3'
Spaces inserted every three bases allow the sequence to be entered as triplet codons. Also, placing
spaces at specified intervals simplifies verification of the sequence. This is for convenience only
and has no effect on the synthesis.
ERASE
Select to delete all entries from the DNA strand being edited.
Upon selecting ERASE, the following display appears:
1 - To erase DNA strand, press yes.
no
yes
menu
YES
Deletes the DNA strand designated in the menu and returns to the previous
menu.
NO
Returns to the previous menu without deleting the sequence.
Section 3: Software Menu Descriptions
3-9
Applied Biosystems
How to Insert a Base
Insertions are made at or to the left of the cursor. For example, to insert T in the third position in the
sequence 5'AAA 3':
5'>AAA
<3'
5'>AATA
<3'
5'>AAAT_
<3'
select T
To insert T in the fourth position in the sequence 5'AAA3':
5'>AAA_
<3'
select T
Continuously pressing the button corresponding to a base, will repeatedly insert that base.
How to Delete a Base
Most deletions are made at the cursor position. For example, to delete C from the sequence
5'AGCT 3':
5'>AGCT
<3'
select delete
5'>AGT
<3'
When the cursor is at the 3'-end of the sequence, the deletion is made to the left of the cursor. For
example, to delete T from the sequence 5'AGCT 3':
5'>AGCT_
<3'
select delete
5'>AGC_
<3'
Multiple bases within parentheses are deleted when the cursor is on either parenthesis. For example,
to delete AGCT from the third position in the sequence 5'AA(AGCT)TTT 3':
5'>AA(AGCT)TTT
<3'
select delete
5'>AATTT
<3'
To erase a single base within parentheses place the cursor on the unwanted base and press delete.
For example, to delete C from the sequence 5'AA(AGCT)TTT 3':
5'>AA(AGCT)TTT <3'
select delete
5'>AA(AGT)TTT
<3'
Continuously pressing delete will repeatedly erase entries.
3-10
Section 3: Software Menu Descriptions
Applied Biosystems
How to Change a Base
To change a base, you first insert the desired base, then delete the unwanted base. For example, to
change 5' AATG 3' to 5' AACG 3':
5'> AATG <3'
select C
5'> AACTG <3'
select delete
5'> AACG <3'
You can enter up to 250 characters (a combination of bases, spaces and parentheses) to create a single sequence. The screen shows a maximum of 28 characters at a time. When additional characters
are entered, the display shifts to the left and the base(s) at the 5' position will no longer be seen. To
observe parts of a sequence not displayed, press the right or left arrow key (→ or ←) to move the
cursor toward the area you want to see.
Note
Once a synthesis has begun, the DNA sequence which is currently being
synthesized cannot be changed.
The DNA Editor Menu can be accessed during an active synthesis and new DNA sequences can be
created for use in subsequent syntheses. Entering a new sequence will not affect the one currently
being made even if the active strand is erased or edited.
The Copy Key
The COPY key is used to transfer an exact duplicate of one DNA strand (e.g. DNA-1) into another
DNA strand (e.g. DNA-2). This feature is useful when performing consecutive syntheses of similar
but not identical sequences. For example, if DNA-1 has a 60 base sequence, the sequence can be
copied into DNA-2. DNA-2 can then be edited instead of having to enter the entire sequence. To
transfer a copy of DNA-1 into DNA-2, the first page of the DNA editor should read:
Select action for
edit
copy
DNA-1
print
60-mer
next
Choose COPY and the menu will state that the sequence in DNA-1 will be copied into another DNA
strand (in this example, DNA-2):
Copy DNA-1 into
enter
Section 3: Software Menu Descriptions
DNA-2
0-mer
next
menu
3-11
Applied Biosystems
If DNA-1 will be copied into a different location, select NEXT until the correct DNA strand is
shown. In this example, DNA-2 has zero bases. However, the copy command will over-write any
DNA sequence currently in DNA-2.
Select ENTER to execute the copy command. The DNA Edit Menu will then be displayed showing
the sequence in DNA-2 and enabling any necessary editing. Note that the DNA sequence will not
only be in DNA-2, but will also remain in DNA-1.
2-5'>AGC AGC AGC AGC AGC AGC AGC <3' 60
(
space
)
erase
menu
The Print Key
The printout from the DNA Editor not only includes the DNA sequence, but also lists the total number of bases, the molecular weight, the base composition, and the time and date. Note that base X
and mixed-base sites are not included in the molecular weight calculation. The following example
shows a DNA Editor printout.
DNA SEQUENCE 1
3-12
NUMBER OF BASES:
BASES USED
DALTONS:
TIME
DATE:
10
A=2
C=2
2644
10:00
06/01/89
5' > AGCTXXAGCT
<3'
G=2
T=2
X=2
Section 3: Software Menu Descriptions
Applied Biosystems
Main Menu Option: Start Synthesis
This section describes the software of the start synthesis menu. For more details on how to begin a
synthesis refer to Section 2: Pre-synthesis Check List and How to Begin a Synthesis.
When the instrument is ready to begin a synthesis, the START SYNTH (synthesis) key is displayed
on Page 1 of the Main Menu. Upon selection, the screen shows the Configure Synthesis Menu. Use
it to choose the DNA strand to be made and the synthesis cycle and ending method to be performed:
DNA-1:12
Make
begin
nxt DNA
Cycle-1:63
nxt cyc
Trityl OFF
ON/OFF
main
The above example shows DNA-1 which has a 12 base sequence (DNA-1:12), Cycle-1 which has
63 steps (Cycle-1:63) and the ending method which is trityl off. Note that the most recently edited
DNA strand and cycle are automatically displayed.
If the menu shows the correct information, synthesis can begin. Any incorrect information must be
changed using the following keys:
NXT DNA
Specifies the subsequent DNA strand. Continue to press this key until the correct DNA strand is displayed.
NXT CYC
Specifies the subsequent synthesis cycle. Continue to press this key until the
correct cycle is displayed.
Note
When the main power is first turned on, the Applied Biosystems ROM cycles are
automatically loaded into the correct RAM cycle locations as shown on the next page.
For example, to synthesize using the .2µM cycle, simply display Cycle-1:63. (You can
create your own cycles by editing a RAM cycle. Once you change a cycle, the edited
version will appear in the Start Synthesis Menu. Refer to The Cycle Editor Menu for
instructions.)
ROM cycle name
RAM Cycle location
Number of steps
.2µM
Cycle-1
63
1µM
Cycle-2
64
Low
Cycle-3
61
10µM
Cycle-4
53
Section 3: Software Menu Descriptions
3-13
Applied Biosystems
ON/OFF
Specifies the desired ending method. When one ending method is shown,
pressing ON/OFF will display the other.
About the Ending Method:
The 5' terminus of the fully synthesized DNA chain can either remain protected by a dimethoxytrityl
group (trityl on), or can be detritylated to yield a 5'-hydroxyl (trityl off). Trityl on is usually selected
when purifying by trityl-specific OPC or reverse phase HPLC. Trityl off is usually chosen when
purifying by gel electrophoresis or ion exchange HPLC.
Note
If trityl on is specified, the DNA can be manually detritylated by a 15-minute treatment
with 80% acetic acid at room temperature. This is done following synthesis after base
deprotection.
When all synthesis information is displayed correctly, press BEGIN. Once pressed, you can no
longer change the DNA strand and the synthesis cycle selection. However, cycle step times can be
edited during synthesis by using the Cycle Editor.
Although the ending method is designated before beginning a run, it can be changed during a synthesis until the final addition cycle is complete. This is done from Page 4 of the Monitor Synthesis
Menu. After the last step of the final base addition, the ending method cannot be changed.
After you select BEGIN, the screen reads:
Checking bottle usage for base 1
Please wait . . .
Each reagent will be checked for usage and for when it will need replacing. According to alarms
that you must set in the Change Bottles Menu, the screen will show if and when the synthesis will
be interrupted by the alarm. For example, if an oligomer 20 bases long will be synthesized, and tetrazole (Bottle 9) will empty after performing 11 cycles of base addition, the screen will read:
Bottle 9 will interrupt in 11 cycles
begin
menu
You then have the option of allowing the alarm to be triggered (interrupting the synthesis after eleven base additions) or changing the bottle immediately. To replace the bottle, return to the Main
Menu and choose the Change Bottles Menu. When bottle usage has been checked and synthesis is
ready to start, press BEGIN.
3-14
Section 3: Software Menu Descriptions
Applied Biosystems
After pressing BEGIN, the display will show which column you need to place on the instrument.
For example, if T is the 3' terminus of DNA-1, the screen will read:
Install (T) Column, then press enter
enter
menu
Follow the instructions and install a (T) column. Be sure to use the column size corresponding to
the synthesis cycle. Remember to record the serial number to identify the synthesis. After installing
the column, select ENTER and the display will read:
Purge Phosphoramidite lines?
no
yes
menu
IMPORTANT You can press the MENU key from any of the above menus to cancel the start of the
synthesis. The display will then show the Configure Synthesis Menu allowing you to
begin again.
The above menu gives the option of purging the tetrazole and phosphoramidite delivery lines prior
to beginning a synthesis. A purge refills these lines with fresh reagents. This is strongly recommended when the instrument has been idle for more than 12 hours (more than 6 hours in humid environments) or if one of the phosphoramidite reservoirs has not been accessed within 12 hours. Over
this time oxygen and atmospheric water can penetrate the delivery lines and cause failures of the
fast addition reaction.
Answer the question, do you want to purge the phosphoramidite delivery lines? Choose YES or NO.
NO
Bypasses the purge steps. Synthesis automatically begins and the menu displaying the synthesizing status is shown. NO is usually chosen when the instrument has been running continuously and all phosphoramidites have been
accessed within 12 hours.
YES
Purges the phosphoramidite lines. The screen then shows the step number being performed and the time remaining for that step:
Phos purge step
seconds
Section 3: Software Menu Descriptions
1
9
of
9
steps.
stop
3-15
Applied Biosystems
When all steps are finished, the synthesis automatically begins and the menu displaying the synthesizing status is shown.
STOP
Ends the purge by performing steps 8 and 9 of the procedure which rinse the
reagent valve blocks.
The following menu is then shown:
Rinsing . . . step
seconds
8
of
9
steps
5
stop
Once you stop the purge, the configure synthesis menu is shown and a new synthesis can begin.
Main Menu Option: Monitor Synthesis
When you select MONITOR SYNTH (synthesis), the instrument status is displayed and one of the
following five menus is shown.
Synthesizing status is shown during an active synthesis:
SYNTHESIZING base x of xx bases
hold
jump
intrpt
more
main
(x = the number of the base currently being coupled as counted from the 3'-terminus, xx = the total
number of bases in the sequence)
Interrupted status is shown when a run has been temporarily stopped mid-synthesis:
INTERRUPTED at base x of xx bases
resume
jump
abort
more
main
Holding status is shown when an individual synthesis step is being executed indefinitely:
HOLDING base x of xx bases
resume
3-16
jump
intrpt
more
main
Section 3: Software Menu Descriptions
Applied Biosystems
Synthesis Complete is shown when a synthesis is finished:
SYNTHESIS COMPLETE
main
Synthesis Aborted is shown after permanently ending a run mid-synthesis:
SYNTHESIS ABORTED
main
Instrument Status: Synthesizing
When you press MONITOR SYNTHESIS during a run, Page 1 of the Synthesizing Status Menu is
displayed:
SYNTHESIZING base x of xx bases
hold
jump
intrpt
more
main
It shows the total number of bases in the sequence (xx) and the number of the base currently being
synthesized as counted from the 3' terminus (x).
IMPORTANT Although the DNA sequence is entered 5' to 3', the DNA chain is synthesized 3' to 5'.
In all menus selected from Monitor Synthesis, the 3' terminus is shown as base
number 1 and subsequent bases are numbered 3' to 5'.
Summary of Synthesizing Status Menu Options
HOLD
When you press HOLD, the current step will be executed indefinitely. See The
Holding Menu for details.
JUMP
Allows you to perform an out-of-sequence step, before or after the current step,
and then continue with the synthesis. See The Jump Step Menu for details.
INTRPT
Allows you to stop a synthesis immediately and then continue it, program a future interrupt or abort a run. See The Interrupt Menu for details.
Section 3: Software Menu Descriptions
3-17
Applied Biosystems
Displays additional information about the synthesis. See below.
MORE
Synthesizing Status Menu options in detail
The MORE key:
Selecting MORE from page 1 of the Synthesizing Status Menu displays Page 2. It shows additional
information about the synthesis including which step and function are being performed, and the total time of duration and time remaining for that step. For example, during step 1 shown below, acetonitrile (bottle # 18) is delivered to waste for a total of 5 seconds with 3 seconds remaining until
completion.
Step:1
hold
#18 TO WASTE
jump
intrpt
3 OF 5 SEC
more
main
Selecting MORE again displays Page 3 which shows the base currently being coupled (as denoted
by the blinking box) and which bases will be subsequently added. For example, in the sequence
5'AAA AAA AAA GCT 3' shown below, C is currently being coupled, G will be added next which
is followed by A and so on. The number in the upper left corner signifies which DNA strand is being
synthesized (for example: 1 =DNA-1).
1-5'>AAA AAA AAA GCT
hold
Note
jump
<3'
intrpt
more
main
The hold, jump and intrpt keys operate from pages 1, 2 and 3 of the synthesizing
status menu.
Selecting MORE again displays Page 4 which shows the DNA strand being synthesized (e.g. DNA1, a 12-base sequence), the cycle being performed (e.g. Cycle-1 which has 63 steps) and the ending
method (e.g. trityl off).
Making
ON/OFF
DNA-1:12
Cycle-1:63
Trityl OFF
more
main
The ON/OFF key operates during synthesis to change the ending method from Trityl OFF to Trityl
ON and vice versa. Following the last step of the final base addition, the ending method cannot be
changed.
3-18
Section 3: Software Menu Descriptions
Applied Biosystems
Selecting MORE again returns to Page 1 of the Synthesizing Status Menu.
The Holding Menu
When you select HOLD, the step currently being executed will continue indefinitely. During a
Hold, all activated valves remain open. The Hold can be used to increase the delivery time of a reagent. The Holding Menu and its relationship to the Synthesizing Status Menu are shown in Figure
3-2. Upon pressing HOLD, the screen will read:
HOLDING base x of xx bases
resume
jump
intrpt
more
main
Select MORE for additional synthesis information. Note that the time remaining for the step freezes
when you press HOLD.
STEP:1
resume
#18 TO WASTE
jump
intrpt
3 OF 5 SEC
more
main
Select MORE again for further information:
1-5'>AAA AAA AAA GCT
resume
jump
<3'
intrpt
more
main
Select MORE again for further information:
Making
ON/OFF
Note
DNA-1:12
Cycle-1:63
Trityl OFF
more
main
The resume, jump and intrpt keys operate from Pages 1, 2 and 3. The ONIOFF key
operates from Page 4.
IMPORTANT When a chemical delivery step is on hold, the extra reagent consumption is not
recorded in bottle usage and is not included in the alarm tabulation.
RESUME
Releases the Hold on the step and continues the synthesis.
Section 3: Software Menu Descriptions
3-19
Applied Biosystems
JUMP
Shows the Jump Step Menu, the hold is not yet released. Using Jump Step, the
synthesis can be continued at any point in the cycle before or after the step currently on Hold. The Hold is released when the jump is activated. Jump Step allows parts of the chemical cycle to be skipped or repeated. Refer to The Jump
Step Menu for further instructions.
INTRPT
Displays the first page of the Interrupt Menu. When you interrupt immediately,
it releases the Hold on the step and stops the synthesis. The synthesis will not
continue until the interrupt is removed. For further instructions, refer to The Interrupt Menu.
MAIN
3-20
Returns to the Main Menu. This will not affect the Holding status.
Section 3: Software Menu Descriptions
Figure 3-2.
The Holding Menu
Applied Biosystems
Section 3: Software Menu Descriptions
3-21
Applied Biosystems
The Jump Step Menu
Using JUMP, the synthesizer can execute any step in the cycle before or after the current step being
performed. Jumping to out-of-sequence steps enables you to repeat or skip parts of the chemical cycle. The jump step menu and its relationship to the Synthesizing Status Menu are shown in Figure
3-3.
When you select JUMP, the step being executed will continue indefinitely, like pressing HOLD.
The screen will display the Jump Step Menu showing the current step, for example, step 1, #18 to
waste:
Enter step #1
enter
#18 TO WASTE
next
prev
menu
Follow the prompt and enter the step number to be performed next. The number and the corresponding function description will appear on the screen, be sure it is correct. Then, press ENTER:
ENTER
Continues synthesis from the specified step. The screen will then display Page
2 of the Synthesizing Status Menu showing the new step being performed. If
you press ENTER without specifying a step number, the synthesis will
continue from the same step.
IMPORTANT Jumping to step 1 increments to the next base. For example, when synthesizing base
2 and you jump to step 1, the synthesis will proceed with step 1 of base 3. To remain
within the base 2 cycle, jump to step 2 or any subsequent step.
During synthesis, if you jump over Cycle Entry, the cycle counter which records bottle
usage will not increment.
NEXT
Displays the subsequent step and its function description. Continue to press
NEXT until the desired step is shown.
PREV
Displays the preceding step and its function description. Continue to press
PREV until the desired step is shown.
MENU
3-22
Upon selecting MENU, a jump will not occur. The step being held will be released, the synthesis will resume and the display will return to the synthesizing
status menu.
Section 3: Software Menu Descriptions
Applied Biosystems
Figure 3-3.
The Jump Step Menu
Section 3: Software Menu Descriptions
3-23
Applied Biosystems
The Interrupt Menu
Selecting INTRPT (interrupt) allows you to stop a run at a specified step mid-synthesis. Using Interrupt, a synthesis can be aborted, stopped immediately and then continued or programmed to stop
at any future step in the synthesis. Once a run has been interrupted, a jump step can also be performed. The set of interrupt menus and their relationship to the Synthesizing Status Menu are shown
in Figure 3-4.
IMPORTANT Interrupt synthesis at a safe step, such as Cycle Entry or Cycle End, so that the DNA
is left chemically stable.
Upon selecting INTERRUPT, one of two menu pages will be displayed. (This does not yet interrupt
the synthesis.)
1. When an interrupt has been programmed to occur in the future, the screen shows when it will
happen. For example:
Interrupt at step 1
ahead
imediat
base 2
clear
menu
2. When an interrupt has not been programmed, the screen reads:
No interrupt set
ahead
Note
imediat
clear
menu
The IMEDIAT (immediate) and MENU keys operate from both pages. AHEAD
only operates from the “no interrupt set” page and CLEAR only from the “interrupt at step_ base_” page.
Interrupt Ahead
AHEAD
After pressing AHEAD from the “no interrupt set” page, the screen displays:
Interrupt at step 0
ahead
imediat
base 0
clear
menu
By choosing AHEAD, you can program an interrupt to occur at a future step anytime during the
synthesis. This is done by entering the step number and base number where the synthesis should
3-24
Section 3: Software Menu Descriptions
Applied Biosystems
stop. If no base is specified, the interrupt will occur at the next base. To enter the values, move the
cursor as necessary and type the desired numbers. For example, to stop the synthesis the moment it
reaches Step 1 of the first addition cycle, enter “1” next to step and “2” next to base. (Remember,
the support-bound nucleotide is base 1. Base 2 is coupled during the first addition cycle.) The screen
will read:
Interrupt at step 1
ahead
imediat
base 2
clear
menu
When the synthesis actually reaches this point, it will stop and the menu displaying the Interrupted
Status will be shown.
Only one interrupt can be set at a time. As soon as one has been programmed, an asterisk (*) will
appear when viewing the Synthesizing and Holding Status Menus (:intrpt*:) which serves as a reminder. Once set, an Interrupt Ahead may be changed by typing over the existing entries.
CLEAR
Erases an interrupt.
MENU
Returns to Page 1 of the Synthesizing Status Menu.
Interrupt Immediate
IMEDIAT
Closes all valves instantly and stops the synthesis. The screen will display the
interrupted Status and will show when the synthesis was stopped. For example:
Interrupted at base 2 of 12 bases
resume
Section 3: Software Menu Descriptions
jump
abort
more
main
3-25
Applied Biosystems
Select MORE for additional information. Note that the time remaining for the current step freezes
when a synthesis is immediately interrupted.
STEP 1
resume
#18 TO WASTE
jump
abort
3 of 5 SEC
more
main
Select MORE for further information:
1-5'>AAA AAA AAA GCT
resume
jump
<3'
abort
more
main
Select MORE for further information:
Making
ON/OFF
DNA-1:12
Cycle- 1:63
Trityl OFF
more
main
To summarize the above example: The synthesis was interrupted (by pressing IMEDIAT) when it
was coupling base number 2 which is C. It was stopped after performing step 1 (#18 to waste) for
2 seconds (3 seconds remain). There are a total of 12 bases in this sequence (DNA-1:12). Cycle-1
is being performed and the ending method is trityl off.
IMPORTANT Interrupt synthesis at a safe step, such as Cycle Entry or Cycle End, so that the DNA
is left chemically stable.
Note
3-26
The resume, jump and abort keys operate from Pages 1, 2 and 3. The ON/OFF key
operates from Page 4.
RESUME
Continues synthesis from the beginning of the interrupted step. The screen then
returns to the Synthesizing Status Menu.
JUMP
Shows the Jump Step Menu allowing you to continue the synthesis before or
after the interrupted step. The synthesis remains interrupted until the jump is
activated. Refer to The Jump Step Menu for instructions.
ABORT
Select to end a synthesis prematurely. This might be done if a sequence was
entered incorrectly. When pressed, the display reads:
Section 3: Software Menu Descriptions
Applied Biosystems
To abort synthesis, press yes.
no
yes
main
Follow the instructions and choose yes or no.
YES
Ends the synthesis permanently. Once you press YES, the synthesis cannot be
resumed. The display will then read:
Synthesis Aborted
main
NO
Prevents aborting the synthesis. The interrupt is not affected and the display returns to the Interrupted Status Menu.
Section 3: Software Menu Descriptions
3-27
Figure 3-4.
The Interrupted Menu
Applied Biosystems
3-28
Section 3: Software Menu Descriptions
Applied Biosystems
Main Menu Option: Change Bottles
The Change Bottles Menu provides a procedure for removing empty reservoirs and replacing them
with bottles of fresh reagents. In addition, it displays bottle usage information and provides an alarm
to alert you that reagent levels are low.
Upon selecting CHANGE BOTTLES, the screen will read:
#1 A-PHOS
next
alarm: 0
prev
change
cycle: 0
main
Menu Description
A reservoir number and an abbreviated description of its contents are displayed. The above example
shows Bottle 1 (#1) which contains A-phosphoramidite. The screen also shows the alarm and cycle
(or bottle usage) information for the reservoir being viewed.
The Cycle Setting
Cycle: displays the number of cycles a reservoir has been accessed. During a synthesis, the software
tabulates which bottles are accessed during each cycle of base addition. Tabulation occurs when
Function 33, cycle entry, has been reached. If a bottle has been used (at least once or more than
once) during that cycle, the cycle counter increases by one. When performing the bottle change procedure, the counter is automatically reset to zero when a bottle is removed and replaced. If a phosphoramidite is partially used, stored frozen and then reused, the cycle counter should be reset.
Simply move the cursor to the right of cycles: and enter up to a 3-digit number.
The Alarm Setting
Alarm: displays the number of cycles which will trigger the alarm. You need to set this number according to the guidelines described in the following pages. (See About the Alarm and Table 3-1,
Bottle Usage Data.) To set the alarm, move the cursor to the right of alarm: and enter up to a 3-digit
number.
Note
If the alarm is set to zero, it will not function.
Section 3: Software Menu Descriptions
3-29
Applied Biosystems
The Change Bottles Menu Keys
NEXT
Displays the cycle and alarm information for the subsequent reservoir. When
bottle 18 is being viewed, selecting NEXT will show bottle 1.
PREV
Displays the cycle and alarm information for the preceding bottle. When bottle
1 is being viewed, selecting PREV (previous) will show bottle 18.
CHANGE
Initiates the steps for the bottle change procedure.
About the Bottle Change Procedure
The bottle change procedure is used to remove empty reservoirs and replace them with bottles of
fresh reagents. It can be done before beginning a synthesis or when an active synthesis has been
interrupted either manually or by the alarm. This process is especially important for preventing oxygen and water contamination of atmosphere-sensitive phosphoramidites and tetrazole.
In summary, the procedure begins by removing old reagent from the delivery line and forcing it
back into the reservoir by an acetonitrile wash. The bottles are then removed and replaced. Next,
argon purges the reservoir’s headspace to eliminate air and the delivery line is then refilled with
fresh reagent. For a complete description of the bottle change procedures, see Section 4.
To begin the procedure, press CHANGE. The screen will read:
Enter bottle # to change: 1
enter
press enter
menu
Follow the instructions and enter the bottle number that you want to change. Note that the screen
will automatically display the bottle number which was showing in the previous menu. If a different
reservoir needs to be changed, type the appropriate number and it will appear on the screen. Be sure
it is the correct number as printed on the bottle label and above its receptacle. Next, press ENTER
and the steps which prepare the bottle for removal will begin.
The screen will display which bottle is being changed, the step number being performed and the
time remaining for that step. For example, if the A-phosphoramidite reservoir is empty and needs
replacing; enter “1” (unless 1 is already displayed), press ENTER and the screen will read:
Changing # 1
A-PHOS
STEP 1 OF 9 STEPS
3-30
10 sec
stop
Section 3: Software Menu Descriptions
Applied Biosystems
Select to halt the current step and return to the previous menu. Stop has the
same function in all Change Bottles displays.
STOP
Note
Do not reuse the phosphoramidites or tetrazole after these steps are completed
because the reservoirs now contain HPLC grade acetonitrile from bottle 18.
If you are going to remove partially used phosphoramidites, store them and reuse
them later; the bottle change procedure should not be performed. See Section 2 for
instructions on How to Store Dissolved Phosphoramidites.
When these steps are completed, the screen displays:
Change bottle 1 A-PHOS
enter
then enter
stop
Follow the prompt, Change Bottle 1 A-PHOS. Remove the empty reservoir and upon opening the
fresh bottle, quickly place it on the instrument. (For details on How to Install Reagent Bottles, refer
to Section 2.) After the reservoir is correctly installed, press ENTER. The reservoir is then prepared
for chemical deliveries and the cycle counter is reset to zero. The screen will now display the step
number being performed and the time remaining for that step.
Changing # 1
A-PHOS
STEP 6 OF 9 STEPS
5 sec
stop
When the procedure is finished, the display returns to the first menu in this series enabling you to
change an additional bottle. To return to the Main Menu, select MAIN. Now the synthesis can either
begin by selecting START SYNTH or continue by releasing the interrupt (select MONITOR
SYNTH and press RESUME), as needed.
About the Alarm
The alarm is triggered when a bottle has been used a specified number of cycles. To operate effectively, the alarm must be set correctly. If set too high, the reservoir could run dry and the synthesis
could fail.
Chemical consumption can vary with each instrument due to different pressure regulator settings
and use of different cycle scales. Bottle usage data is provided in Table 3-1 as a guideline for setting
the alarm. This data shows the average number of uses (or cycles) obtainable per bottle. When ini-
Section 3: Software Menu Descriptions
3-31
Applied Biosystems
tially setting the alarm, use values which are 5 to 7 uses less than these numbers and adjust as necessary.
IMPORTANT Since consumption rates will vary from instrument to instrument, it is important to
visually monitor reagent consumption for the first several syntheses. Once you
determine the usage, the alarms can be effectively set.
Note that the number of uses obtainable per bottle will change depending on which cycle is being
performed (e.g., .2µM, lµM, Low or 10µM). Longer chemical delivery steps result in greater reagent consumption and fewer uses per bottle. Consider these factors when setting the alarm.
Phosphoramidites used during the phosphoramidite purge procedure are not tabulated in the cycle
counter. This process uses less than one half the amount of phosphoramidite used in a coupling step
of a one-micromole synthesis. In addition, manual deliveries (using the Manual Control Menu), deliveries during a hold step, and acetonitrile used during the bottle change procedure are not tabulated in the cycle counter. When synthesizing mixed-sequence probes, each phosphoramidite which is
delivered is tabulated in the cycle counter.
When the alarm is triggered, the instrument will finish the current addition cycle, interrupt the synthesis at Cycle Entry and sound an alarm. A blinking “A” will be shown in the bottom right corner
of any menu being viewed. Press the MENU key until the display reads:
Bottle Empty Alarm!!!
Change bottle to clear alarm.
main
Select MAIN to return to the Main Menu and then select CHANGE BOTTLES. Once the Change
Bottles Menu is showing, the alarm message will be cleared. However, the synthesis will still remain interrupted. Proceed with the bottle change procedure as described earlier in the section. When
finished, return to the Main Menu, select MONITOR SYNTH and continue the synthesis by selecting RESUME.
If the alarm has been triggered but the bottle is not yet empty, the synthesis can be continued by
clearing the interrupt (by choosing MONITOR SYNTH and then RESUME). The alarm message
can be cleared by simply viewing the Change Bottles Menu. If this is done, it is important to reset
the alarm to an appropriate number.
3-32
Section 3: Software Menu Descriptions
Applied Biosystems
Bottle Usage Data
Table 3-1. Bottle Usage Data
Synthesis Scale
0.2µM
1.0µM
(cycles)
(cycles)
Reagent
Phosphoramidites
0.25 grams: A
G
C
T
Low
(cycles)
10.0µM
(cycles)
18
18
18
20.7
12.5
12
12.3
13.7
28
27
28
31
2
2
2
2.25
0.5 grams
A
G
C
T
37.5
36.5
37
41.5
25
24
24.5
27.5
56
54.5
55.5
62
4
4
4
4.5
1.0 grams
A
G
C
T
75
73
74
83
50
48
49
55
112
109
111
124
8
8
8
9
Tetrazole (180 mL)
411
343
561
101
Acetic anhydride (180 mL)
732
610
732
183
NMI (180 mL)
TCA (450 mL)
732
190
610
190
732
190
183
74
Iodine (200 mL)
486
486
486
119
Acetonitrile (4 L)
803
803
803
156
Section 3: Software Menu Descriptions
3-33
Applied Biosystems
Main Menu Option: Cycle Editor
Introduction
Use the Cycle Editor to create, edit and print synthesis cycles. Four fully programmable, synthesis
cycle locations are available in RAM (Random Access Memory). They are named Cycle-1 through
Cycle-4. RAM cycles can be created or edited according to your needs. Immediately before synthesis, you select the RAM cycle you want to use from the Start Synth Menu.
Applied Biosystems also supplies four cycles which are permanently stored in ROM (Read Only
Memory). These ROM cycles are optimized for synthesizing on the .2-micromole, one-micromole
and ten-micromole scales. They are named .2µM, 1µM and 10µM respectively. The fourth cycle is
named Low. It is a low reagent-consumption cycle on the .2-micromole scale which uses 33% less
phosphoramidites. Cycles are further explained in Section 4.
To synthesize using a ROM cycle, it must be transferred to a programmable RAM cycle location
(i.e. Cycle-1, 2, 3 or 4). Only the RAM cycles appear as choices in the Start Synthesis Menu; .2µM,
1µM, Low and 10µM cycles do not appear as options.
Note
The first time the main power is turned on, the Applied Biosystems ROM cycles are
automatically loaded into the RAM cycle locations as shown below. For example, to
synthesize using the .2µM cycle, simply display Cycle-1:63 in the Start Synthesis
Menu.
ROM
synthesis
cycle
RAM
cycle
location
Total
Number
of steps
cycle
time
(min.)
crude
yield* (O.D.)
(20mer)
.2µM
Cycle-1
63
5.5
20-25
1µM
Cycle-2
64
5.5
100-120
Low**
Cycle-3
61
5.5
20-25
10µM
Cycle-4
53
24
800-1000
*Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming 33
micrograms/O.D. unit. **The Low cycle is on the .2µM scale.
After the ROM cycles are transferred to RAM cycle locations, they will be stored there indefinitely
until you change or delete them. Once transferred, they can be edited as desired. Once you edit a
cycle, the edited version will be stored in RAM and will appear in the Start Synthesis Menu. If the
3-34
Section 3: Software Menu Descriptions
Applied Biosystems
main power is turned off, the edited version will still be stored in RAM. If you want to transfer a
ROM cycle back into a RAM location, use the COPY key.
The Cycle Editor Menu
Upon selecting CYCLE EDITOR, the display shows a cycle name and the number of steps in that
cycle. If a cycle has zero steps, it has not been defined. The following example shows Cycle-1 which
has 63 steps due to the automatic transfer of the .2µM cycle:
Select action for:
edit
Cycle- 1:63 steps
copy
print
next
main
Follow the prompt and select an action for Cycle-1.
EDIT
Select to create, modify or review the steps of the designated cycle. The EDIT
key will not function when viewing the ROM cycles (.2µM, 1µM, Low,
10µM). To edit these cycles, transfer them to a programmable, RAM cycle location using the COPY key.
COPY
Select to duplicate the designated cycle into any programmable, RAM cycle
location (1, 2, 3 or 4).
PRINT
Select to print the designated cycle.
NEXT
Select to view the subsequent cycle name and the number of steps in that cycle.
Press NEXT until the desired cycle is shown. The cycles will appear in the following order, .2µM, 1µM, Low, l0µM, Cycle-1, Cycle-2, Cycle -3, Cycle-4.
The Edit Key
Upon selecting EDIT, the screen will display step zero and the total number of steps in the cycle:
C1- 0 of 63 steps in Cycle- 1
next
Section 3: Software Menu Descriptions
prev
insert
delete
menu
3-35
Applied Biosystems
An abbreviated cycle name appears on the top left of the display to signify the cycle being viewed
(C1 = Cycle-1). Step zero (-0) is not an active step and is not performed during synthesis. Because
all new steps are added after existing ones, it appears so that step 1 can be inserted, if necessary.
NEXT
Select to view the subsequent step. When viewing the last step, pressing NEXT
will show step zero.
PREV
Select to view the preceding step. When viewing step zero, pressing PREV
(previous) will show the last step.
INSERT
Select to insert a step in an existing cycle or to enter steps when creating new
cycles.
DELETE
Erases the step that is currently showing.
IMPORTANT All steps in the cycle are erased by selecting DELETE when step “0” is showing.
When DELETE is chosen, a menu confirming the action appears:
Press yes to delete all steps in Cycle-1
yes
menu
Press YES to erase the cycle. Press MENU to avoid erasure and to return to the previous menu.
Editing an Existing Cycle
When viewing step zero of an existing cycle, press NEXT and step 1 appears. It shows the function
number and description, and the step time. In addition, it shows that the step will be active for the
bases which are listed.
C1-1
next
Fxn:10
prev
#18 TO WASTE
insert
t=2
delete
AGCTX
menu
The above display shows Cycle-1 (C1), step 1 (-1) and function 10 (Fxn:10) which is #18 TO
WASTE. The step will occur for 2 seconds (t=2) and will be performed when synthesizing all bases
(AGCTX).
3-36
Section 3: Software Menu Descriptions
Applied Biosystems
When viewing this menu, the current step can be edited by changing the function, the time and the
bases. To view another step in the cycle, place the cursor under the current step number, type the
new number and two new menu options (JUMP and UNDO) will be shown. Press JUMP and the
screen will show the information pertaining to the new step. Press UNDO to return to the original
step. NEXT and PREV can also be used to view subsequent and preceding steps.
To change the function, move the cursor under the existing function number and type the desired
number over it. The function description corresponding to the new number will automatically be
displayed.
Similarly, to change the step time, move the cursor to the existing time and type a new one over it.
Time is displayed in seconds with a maximum entry of 999 seconds. The function number and time
can also be erased by placing the cursor under the existing number and pressing the gray delete key
on the fixed keyboard.
The Base Specifier Field
When a base is listed in the base specifier field (located in the top right corner of the display), the
step will be active when synthesizing that base. Since most syntheses are performed so that all steps
are active for all bases, every base (AGCTX) is automatically listed for each step and therefore
needs no adjustment. However, to execute a step for only certain bases, the unwanted bases must
be deleted from the base specifies field. To indicate that a step should not be performed for a particular base, first move the cursor to the unwanted base (e.g. X). When the cursor is under a base,
two new menu options, YES and NO, appear:
C1-1
next
Fxn:10
prev
#18 TO WASTE
insert
t=2
yes
AGCTX
no
NO
Erases the specified base and the step will not occur when synthesizing that
base.
YES
If a base has already been erased, move the cursor to the base position and select YES. The base will then reappear and the step will occur when synthesizing that base.
To display the DELETE and MENU keys again, move the cursor under the time, step or function
number.
Section 3: Software Menu Descriptions
3-37
Applied Biosystems
Creating a New Cycle/How to Insert Steps
The INSERT key is used to add steps to existing cycles and to create entirely new cycles. To create
a new cycle, begin by viewing step zero of the desired programmable, RAM cycle (e.g. Cycle-4):
C4-0 of 0 steps in Cycle- 4
next
prev
insert
delete
menu
IMPORTANT All steps of an existing cycle will be erased by pressing DELETE while observing step
zero.
Select INSERT and the display will read:
C4-1
step ok
Fxn:_
AGCTX
exit
To define the first step, move the cursor next to Fxn:_, and enter the correct function number. The
corresponding function description will automatically appear. If an invalid function is entered, Bad
Function is displayed. Once a function has been entered, the symbol for the step time (t=) appears.
Move the cursor next to t= and enter the desired time. If the step should occur for every base, all
bases should be showing in the base specifier field. Once you enter the correct information, press
either EXIT or STEP OK.
STEP OK
Select to enter the step in the cycle and to remain in the insert mode. The subsequent step can then be defined and entered. Choose STEP OK when inserting
several consecutive steps.
EXIT
Inserts the step in the cycle and returns to the previous menu. Choose EXIT after completing all insertions or when inserting a single step. If neither a valid
function nor the time has been entered, pressing EXIT will return to the previous menu without inserting the step.
How to Correct Mistakes
To correct mistakes, type over the incorrect entry or erase it by pressing the delete key on the fixed
keyboard. Once a step has been inserted, it can only be erased by viewing the step and selecting the
DELETE soft key.
3-38
Section 3: Software Menu Descriptions
Applied Biosystems
IMPORTANT When creating new cycles, they must contain Function 33, Cycle Entry, and Function
34, Cycle End, for non-zero times (e.g., 1 second) In addition, Cycle Entry should
appear before Cycle End.
Adding a step to an existing cycle is done in the same manner as just described. Note that all new
steps are inserted after existing ones. For example, when step 8 is shown and insert is selected, the
new step will become step 9. All subsequent steps are renumbered accordingly.
IMPORTANT During an active synthesis, only step times can be edited. Any new times will be
effective immediately and will be used during the current synthesis as well as in
subsequent syntheses.
The Copy Key
The COPY key is used to transfer an exact duplicate of one cycle (1, 2, 3, 4, .2µM, 1µM, Low or
l0µM) into a programmable RAM cycle (1, 2, 3 or 4). For example, to modify the .2µM cycle, select
NEXT from the first page of the Cycle Editor until the menu reads:
Select action for: Cycle .2µM 63 steps
edit
copy
print
next
main
Choose COPY and the message will state that the .2µM cycle will be copied into another cycle location (in this example Cycle-2):
Copy Cycle .2µM to:
Cycle-2: 0 steps
enter
next
menu
If .2µM should be copied into a different location, press NEXT until the desired cycle is shown. In
this example, Cycle-2 has zero steps. However, if Cycle-2 were already defined, it would not need
erasing prior to copying. Copying will overwrite any cycle. Select ENTER to execute the copy command. The editor will then be displayed showing step zero of the new Cycle-2 and enabling any
changes.
C2-0
of
next
Section 3: Software Menu Descriptions
63
steps in Cycle-2
prev
insert
delete
menu
3-39
Applied Biosystems
Main Menu Option: Manual Control
Use the Manual Control Menu to manually activate functions and to open and close individual
valves.
IMPORTANT Before opening any valves, refer to the DNA synthesizer schematic or the plumbing
diagram (Appendix B) and the function list (Appendix A) to be sure the correct flow
path will be formed. Also refer to Section 4: Functions, Cycles and Procedures, and
Figure 4-4.
Manual control will not operate during a synthesis unless you interrupt the run.
Upon selecting MANUAL CONTROL, the screen will display:
ON:
valve
VALVE
FXN
ALL OFF
fxn
all off
main
Select to activate a valve.
Select to activate a function.
Immediately closes all valves.
The Valve Key
To open or close valves, first press VALVE and the screen will display:
Enter valve #_
on
3-40
and press on or off.
off
ON
Opens the valve.
OFF
Closes the valve.
menu
Section 3: Software Menu Descriptions
Applied Biosystems
MENU
Erases a valve number after it was entered and returns to the previous menu.
Press MENU if VALVE was accidently selected from the previous display.
To activate the valve, follow the instructions on the screen and enter a valve number. To erase a
mistake, without returning to the previous menu, move the cursor to the unwanted number and press
the delete key on the fixed keyboard.
Next, follow the prompt and press ON or OFF. When you select ON, the valve is activated. The
screen returns to the previous menu and shows the activated valve.
ON: 1
valve
fxn
all off
main
A maximum of eight valves can be opened simultaneously by repeating this procedure (i.e., Press
VALVE, enter the valve number, then press ON).
Select ALL OFF to immediately close all valves. To close a single valve, select VALVE, enter the
appropriate valve number and press OFF. The screen will then display the previous menu and show
the valves still remaining open.
The Function Key
Upon choosing FXN (function), the screen will display:
Fxn # ? _
on
press on or off
off
next
prev
menu
ON
Choose to activate the function.
OFF
Select to deactivate the function and return to the previous menu.
NEXT
Shows the subsequent function number and its description. When the last function is being shown, selecting NEXT will display Function 1.
PREV
Shows the preceding function number and its description. When Function 1 is
being shown, selecting PREV will display the last function.
Section 3: Software Menu Descriptions
3-41
Applied Biosystems
MENU
Deletes the designated function number, closes all valves if a function was activated and returns to the previous menu.
Follow the prompt, FXN # ?, and type a function number. The number and its description will be
shown. For example, when 9 is entered, Function 9, #18 to column is displayed:
Fxn # 9
on
#18 TO COLM
off
next
press on or off
prev
menu
If you enter an invalid function number, that area of the screen will be blank. Next, press ON or
OFF. When you choose ON, the function is activated and the screen returns to the previous menu
which shows the active function. The function can be deactivated by pressing ALL OFF or OFF.
Also, a valve or function will be deactivated automatically upon returning to the Main Menu.
Only one function can operate at a time. When a second one is activated, the first is automatically
deactivated. However, when a function is on, up to eight additional individual valves may be
opened.
Main Menu Option: Fract Pulse
Use the Fract Pulse Menu (fraction collector pulse) to change the pulse time which advances the
fraction collector. When selected, the screen reads:
Fraction collector pulse time: 100 msec
main
Applied Biosystems uses a standard pulse time of 100 milliseconds. You can change the time by
moving the cursor to the unwanted number and inserting a new one over it. When finished, return
to the Main Menu by selecting MAIN.
Main Menu Option: Procedure Editor
Use the Proc (procedure) Editor Menu to create, edit and print the phosphoramidite purge, shut
down and bottle change procedures. Standard procedures, further described in this section, are automatically used during instrument operation. You can, however, edit standard procedures or create
new ones. Note that the flow test procedure, which is accessed from self test, cannot be edited or
printed.
3-42
Section 3: Software Menu Descriptions
Applied Biosystems
When you select PROC EDITOR, the display shows an abbreviated name for the phosphoramidite
purge procedure, #PP PROS PURGE:
Edit/print procedure: #PP PHOS PURGE
edit
copy
print
next
main
Select NEXT to view the subsequent procedure name. The display will then show #1 BOTTLE
CHANGE signifying the bottle change procedure for reservoir 1 which contains A-phosphoramidite. Continuing to press NEXT will show the names for all eleven bottle change procedures and then
will display #SD SHUT DOWN signifying the shut down procedure.
Press NEXT until the desired procedure is showing. Select an action (edit, copy or print) for the
designated procedure.
The Edit Key
Select this option to edit standard procedures or to create new ones. When chosen, the screen shows
step zero of the designated procedure:
PP-0 of 9 steps in PHOS PURGE
next
prev
insert
delete
menu
The Procedure Editor operates in the same way as the Cycle Editor. Refer to The Cycle Editor Menu
for complete instructions.
Once you change a standard procedure, the new user version will automatically be used during subsequent operations and will appear in the editor. To return to the standard version, copy it into the
editor using the COPY key.
The Copy Key
Select this option to copy the designated standard procedure into the editor. Copying is only necessary to restore the standard procedure once it has been edited or deleted. When you press COPY,
the screen reads:
Copy standard version to: #PP PHOS PURGE
yes
Section 3: Software Menu Descriptions
menu
3-43
Applied Biosystems
Choose YES to execute the copy command. Step zero of the procedure will then be shown in the
editor. Once the standard version is restored, it will automatically be used during subsequent operations.
The Print Key
When a printer is connected to the instrument, select this option to print the designated procedure.
When you choose PRINT, the display reads:
Select print version: #PP PHOS PURGE
user
USER
STD
std
menu
Prints the procedure currently in the editor.
Prints the designated standard procedure.
If the standard procedure has not been edited, it will print when you press USER or STD.
Main Menu Option: FXN Editor
Use the FXN (function) Editor to create and edit user functions and to print lists of functions.
You can create new functions not already in memory. These user functions are defined by specifying a set of valves to be opened when the function is activated. Up to eight user functions, called F
92 USER A through F 99 USER H, can be created. Like standard functions, they can be activated
from Manual Control, inserted into any synthesis cycle via the Cycle Editor and into any procedure
via Procedure Editor.
Note
Refer to the DNA Synthesizer Schematic (Appendix B) when creating user functions
and check that the correct flow path will be created. Also refer to Section 4:
Functions, Cycles and Procedures, and Figure 4-4.
The Function Editor is also used to print the standard, test and user functions. Standard functions
include F 1 through 90, 108 and 109 and are used in the synthesis cycle, the phosphoramidite purge,
the bottle change and the shut down procedures. Test functions include F 101 through F 107 and are
used to perform the flow test. Note that the standard and test functions are stored in the permanent
memory and cannot be edited.
3-44
Section 3: Software Menu Descriptions
Applied Biosystems
Upon selecting FXN EDITOR from Page 2 of the Main Menu, the display will read:
Select action for function(s):
edit
print
main
Follow the prompt and select an action:
Choose to create and modify user functions.
EDIT
Prints the complete list of standard, user and test functions when a printer is
connected to the instrument.
PRINT
The Edit Key
When you press EDIT, the screen displays the first user function which is F 92 User A:
92 User A:
next
#_ on
# off
all off
menu
NEXT
Shows the subsequent user function.
# ON
Enters a specified valve in the function. For instructions, refer to the example
below.
Note
The “# on” key enters the valve number but does not activate the valve.
# OFF
ALL OFF
Deletes a valve number from the function.
Deletes all valves listed for the function.
Example
To create a function which would open valves 1, 13 and 17, first press the gray key labeled 1. The
screen will then display the valve number in both the “# on” and “# off” keys:
Section 3: Software Menu Descriptions
3-45
Applied Biosystems
92 User A:
next
#1_ on
#1 off
all off
menu
Next, press the “#1_ on” key and the specified valve number will be entered and shown on the top
line of the display (92 User A: 1). Continue to enter all the valves to be activated by this function,
up to a maximum of 10 valves.
To delete a valve from the function, type the valve number (e.g., 1) and press “#1 off”. To clear all
valves simultaneously, simply select the ALL OFF key.
The Print Key
Upon selecting PRINT, the screen will display:
Select functions to print:
std
user
test
menu
Follow the prompt and select an option:
STD
3-46
Prints the standard functions, F 1 through F 90.
USER
Prints the user functions, F 92 User A through F 99 User H.
TEST
Prints the test functions, F 101 through F 107. Note that F108 and F109 are
printed as test functions, but are used in cycles and procedures.
Section 3: Software Menu Descriptions
Applied Biosystems
Main Menu Option: Power Fail
The Model 391 automatically resumes synthesis after a power failure. Using the Power Fail Menu,
You can program the instrument to remain interrupted and therefore not resume synthesis following
a power outage.
When a power failure occurs during an active synthesis, the synthesis will be interrupted immediately. The instrument cannot operate until the main power returns. During the outage, all synthesis
parameters are retained in memory. These parameters include the DNA sequences; user defined cycles, procedures and functions; bottle usage information and the time.
A maximum power fail time can be set so that if a failure occurs for less than the specified time, the
synthesis will automatically resume when the main power returns. If a failure occurs for longer than
the specified time, the synthesis will remain interrupted and can only continue when you clear the
interrupt.
After selecting POWER FAIL, the screen will read:
Enter maximum power fail time: 0 min
(0 means always continue)
main
To enter the maximum power fail time, move the cursor as necessary and insert the desired value
(up to a 3-digit number). Mistakes can be corrected by typing over an incorrect number.
Note
When zero is entered, the synthesis will always continue when the main power
returns regardless of the duration of the outage.
Example 1:
If the maximum time is 20 minutes and a power failure occurs for less than that, an alarm will sound
for 15 seconds when the main power resumes. The synthesis will then automatically continue and
the following menu will appear.
Applied Biosystems 391 DNA Synthesizer
PCR-MATE EP
Note
Ver. 1.00
start A
When the instrument is not synthesizing, another menu may appear.
Section 3: Software Menu Descriptions
3-47
Applied Biosystems
The blinking “A” in the bottom right corner signifies the alarm has been triggered. To stop the ringing, if necessary, and to view more information, select START and the display will read:
Down Time
7/08
14:20:34 POWER FAIL!!
Up Time
7/08
15:45:44
main A
The down time is the time the power failed. The up time is the time the main power resumed. Select
MAIN to clear this display and return to the Main Menu. If desired, select Monitor Synth from the
Main Menu to view the Synthesizing Status Menu and information about the current step being performed.
Example 2:
If the maximum power fail time is 20 minutes and a power failure has occurred for longer than that,
the synthesis will remain interrupted when the main power returns. The alarm will sound and the
screen will show the two menus mentioned in Example l. However, you must decide whether to resume or abort the synthesis. This is done by selecting MONITOR SYNTH to view the Interrupted
Menu and then pressing RESUME or ABORT.
It is helpful to view page 2 of the Interrupted Menu to determine which step was interrupted. For
example, if the synthesis was stopped during detritylation, the oligonucleotides would be exposed
to acid from the time the power failed. This could cause depurination resulting in some intrachain
cleavage during base deprotection. If the exposure was for several hours, it would be best to abort
the synthesis and begin another one. Note that if you abort synthesis at a step other than Cycle Entry
or Circle End, the valve blocks and delivery lines must be rinsed. Refer to Section 2: How to Abort
a Synthesis, for details.
Main Menu Option: Self Test
Use the Self Test Menu to verify the correct operation of the electronic components of the instrument and to access the flow test procedure. Self test should be performed routinely, approximately
once every few months. In addition, it should be done if the instrument malfunctions.
The easiest way to perform a self test is by pressing the option ALL. If the test passes, the top line
of the display will read PASSED. If a FAILED message is shown, contact the Applied Biosystems
Technical Support Department. Upon choosing SELF TEST, the screen will read:
Select a test . . .
all
3-48
battery
Version 1.00
valves
more
main
Section 3: Software Menu Descriptions
Applied Biosystems
ALL
BATTERY
VALVES
Performs the self test on the battery, the valves, the relays, ROM (read only
memory) and RAM (random access memory). When testing these components,
it is simpler to press ALL instead of each option individually.
Tests for the proper functioning of the battery.
Tests for the proper operation of all valves.
Select MORE for additional test options:
Select a test . . .
relays
RELAYS
ROM
Version 1.00
RAM
more
main
Tests for the proper operation of the fraction collector relays.
ROM
Tests for the proper operation of the permanent memory or the Read Only
Memory.
RAM
Tests for the proper operation of the Random Access Memory or the user defined memory.
Select MORE for additional test options:
Select a test . . .tr.235
keys
display
Version 1.00
clock
more
main
Select MORE for additional test options:
Select a test . . .
tones
repeat
Version 1.00
RESET
more
main
The KEYS, DISPLAYS, CLOCK and TONES key are only used during manufacturing.
REPEAT
Section 3: Software Menu Descriptions
3-49
Applied Biosystems
Repeats the self test for the battery, valves, relays, ROM and RAM until the STOP key is pressed.
RESET
Clears the clock setting and RAM including any stored DNA sequences and
user defined cycles, procedures and functions. After pressing RESET, the
ROM cycles are reloaded into the RAM cycle locations.
When you press RESET, a menu confirming the action appears:
RESET erases ALL user entries/edits
and resets the time --->
RESET
menu
Press RESET to clear all RAM. Press MENU to retain all user memory and to return to the previous
menu.
Select MORE from page 4 of the Self Test Menu to view an additional test option:
Select a test . . .
Version 1.00
flowtst
more
main
Flow Test
FLOWTST
Choose to perform the flow test procedure which measures flow rates through
all essential delivery lines.
Before doing this procedure, refer to Section 2: How to Perform the Flow Test Procedure, for complete details. When you choose FLOWTST, the following display is shown:
Remove all bottles except #18 before you
begin flowtest
begin
more
main
Follow the instructions and remove all the reagent bottles except bottle 18, acetonitrile. Be sure to
properly reseal any chemicals that you will reuse. Press BEGIN and the flow test procedure will
automatically start. The display will then show the current step being performed and its corresponding time:
Step: 1
hold
3-50
WAIT
jump
intrpt
5 of 5 sec
more
main
Section 3: Software Menu Descriptions
Applied Biosystems
This menu is identical to Monitor Synthesis. For details on how to use the HOLD, JUMP and INTRPT keys, refer to the Monitor Synthesis Menu found earlier in this section. Note that the MORE
key does not operate from this menu.
While performing the flow test, you can access the Main Menu by pressing MAIN. To view the flow
test procedure again, select MONITOR SYNTH from the Main Menu.
Steps 1 through 14 sequentially rinse and prime the lines for each bottle position. Use a beaker to
collect the acetonitrile rinse starting with position 15 and working backwards. At step 14 the procedure is interrupted and the screen reads:
Step: 14
INTERRUPT
resume
jump
abort
1 of 1 sec
more
main
The RESUME, JUMP and ABORT keys operate as they do in Monitor Synthesis. This pause is used
to prepare for steps 15 through 43. At this time, place bottles filled with acetonitrile on all positions.
When ready, press RESUME to continue the procedure. Carefully measure the flow from each bottle beginning with position 1, Adenosine phosphoramidite.
After the final measurement, the procedure is interrupted at step 43. Use this pause to prepare to
measure the flow from bottle 14 through the trityl collection line. When ready, press RESUME and
collect the flow.
At step 46, the procedure is interrupted again. The last series of steps flushes the delivery lines with
argon. When ready, press RESUME. Once completed, chemicals can be reattached using the
Change Bottles Menu and synthesis can begin.
Main Menu Option: Set Clock
Use the Set Clock Menu to enter the current time and date. The time is used to record when a power
failure occurred and to provide dates on printouts. After selecting SET CLOCK, the display will
show:
Time:
:
Date:
/ /
main
Move the cursor to the right of time: and enter the current time, then to the right of date: and enter
the date. The date is entered as month/day/last two digits of the year. For example, if the time is ten
A.M. on July 8, 1989, the display should read:
Section 3: Software Menu Descriptions
3-51
Applied Biosystems
Time:
10:00
Date:
07/08/89
main
To correct mistakes, move the cursor to the incorrect entry and type a new one over it.
Note
The clock will continue to update the time, although the time shown on the menu will
remain fixed.
Main Menu Option: Shut Down
Use this menu to prepare the instrument for long term storage. The shut down procedure removes
all reagents in the delivery lines and washes and dries all chemical pathways. The procedure takes
about 17 minutes and is further described in Section 4.
Upon selecting SHUT DOWN, the screen reads:
Replace 9, 11, 12, 14, 15 with empty bottles
enter
main
Follow the prompt and remove bottles 9, 11, 12, 14 and 15 and replace them with clean, empty bottles. Removing the reservoirs prevents HPLC grade acetonitrile from contaminating reagents which
could be reused at a later date. If the phosphoramidites will be reused, their reservoirs should be
removed as well. When finished, press ENTER and the display will show the step number being
performed and the time remaining for that step:
Shutdown
step 1 of 29 steps
seconds: 60
-total time= 17 min.
stop
During these steps, old reagents are removed from the lines by acetonitrile washes. The lines are
then flushed dry. When all steps are completed, the screen will read:
Remove all bottles, wipe all lines.
enter
3-52
stop
Section 3: Software Menu Descriptions
Applied Biosystems
Follow the prompt and take all bottles off the instrument and the wipe all delivery lines with a lintfree tissue. When finished, press ENTER. The screen will then show the steps being performed (additional argon flushes):
Shutdown step x
of 29 steps
seconds:
-total time= 17 min.
stop
When finished, the screen will read:
Place clean bottles on all positions
main
To protect the exposed delivery lines, place clean, empty bottles on all positions.
STOP
Halts the procedure.
Section 3: Software Menu Descriptions
3-53
Section 4:
Functions, Cycles and Procedures
This section contains information essential to understanding functions, synthesis cycles and procedures. Be sure to read the Introduction, Valves, Functions (pages 4-5 to page 4-6) and Synthesis Cycles (pages 4-18 to 4-20). The rest of the section can be used as needed.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2
Valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2
Functions
Synthesis Cycle Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-10
Procedure Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-15
Test Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-18
Synthesis Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-18
Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23
The Phosphoramidite Purge Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23
The Bottle Change Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-24
The Shut Down Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-27
The Flow Test Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-28
Applied Biosystems
Introduction
Automated DNA synthesis requires chemical deliveries to specified destinations on the instrument,
such as the column. These deliveries are controlled by electrically activating solenoid valves to
open and close creating various pathways through the Model 391.
Each solenoid valve is assigned a number which can be used to open or close it. A valve or set of
valves opened or closed simultaneously to perform a specific delivery or task is a function. For example, function 9, #18 to column, delivers acetonitrile to the column. Function 9 opens valves 1,
12, 16 and 17 simultaneously.
A function which is programmed to occur for a specified amount of time is a step. (e.g., Function 9
can be activated for 10 seconds.) A series of steps programmed to perform a chemical process repeatedly is a cycle. Synthesis cycles contain all the steps necessary for the efficient addition of one
base. The cycle is repeated until every base in the sequence is added. Procedures are a series of steps
programmed to complete a task, such as changing a reagent bottle. Upon command, the steps are
performed once and are not repeated. There are four procedures: bottle change, phosphoramidite
purge, shut down and flow test.
Valves
The Model 391 has 24 solenoid valves (numbered 0 to 23) which are opened and closed electrically
and are controlled through the microprocessor. Refer to Section 5: Delivery Valve Blocks, for a
physical description of the valves.
During a synthesis, valves are automatically opened to create the correct chemical pathways and
allow all necessary reagent and gas deliveries. You can also manually operate individual valves by
using the Manual Control Menu. This is helpful when troubleshooting and can be done before synthesis or when a synthesis is interrupted.
All valves with descriptions of what they control are listed next. Refer to Figure 4-1. DNA Synthesizer Schematic, to view the placement of the valves. Valves 0 to 11 control the flow of the following reagents (or argon) from their reservoirs to the reagent valve blocks:
4-2
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Valve:
0
1
2
3
4
5
6
7
8
9
10
11
Controls the flow of:
argon
acetonitrile (bottle 18)
iodine (bottle 15)
TCA (bottle 14)
NMI (bottle 12)
acetic anhydride (bottle 11)
tetrazole (bottle 9)
contents of reserve 5
T-phosphoramidite (bottle 4)
C-phosphoramidite (bottle 3)
G-phosphoramidite (bottle 2)
A-phosphoramidite (bottle 1)
Valve:
12
13
14
15
16
Description:
Directs flow between the reagent valve blocks and the column
Directs flow from the reagent valve block to the waste bottle
Controls the flow of argon to the column valve block
Directs flow from the column valve block to the trityl collection port
Directs flow from the column valve block to the waste bottle
Valves 17-20, 22, 23 are pressure valves which control the flow of argon to the following reagent
reservoirs:
Valve:
17
18
19
20
22
23
Argon to:
acetonitrile (bottle 18)
iodine (bottle 15)
TCA (bottle 14)
all phosphoramidite reservoirs simultaneously
NMI and acetic anhydride (bottles 12 and 11) simultaneously
tetrazole (bottle 9)
Valve
21
Description
Vents all phosphoramidite reservoirs simultaneously
Note that for flow to actually occur, several valves must be opened simultaneously as explained in
the function descriptions, next.
Section 4: Functions, Cycles and Procedures
4-3
Applied Biosystems
Figure 4-1.
4-4
DNA Synthesizer Schematic
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Functions
A valve or set of valves opened simultaneously to perform a specific delivery or task is a function.
Functions 1 through 90, 108 and 109 provide all necessary operations for synthesis. Seven test functions, 101 to 107, are used during the flow test procedure. In addition, up to eight user functions,
numbered 92 to 99, can be created using the Function Editor Menu. Refer to Figure 4-3 for a complete list of all functions.
Functions are automatically performed during a synthesis and require no intervention. When a function is activated, the correct valves automatically open. After a specified time, the function is deactivated and the valves close. Using the Manual Control Menu, functions can also be manually
activated prior to synthesis or when a synthesis is interrupted. This can be useful when troubleshooting.
Functions are given abbreviated names to describe the action they perform. For example, Function
9 (F9) is named “#18 to column”. It delivers the contents of bottle #18, acetonitrile, to the column.
Functions can be grouped according to their action (see Figure 4-4). Note that a single function may
appear in more than one category. Many of the functions are used during the synthesis cycle and the
remainder are used during the bottle change, phosphoramidite purge, shut down and flow test procedures.
During synthesis, you can check which function is being performed by viewing the Monitor Synthesis Menu.
DNA synthesis requires various reagents to be delivered to a specified destination. A reagent can
flow from its reservoir to the waste bottle, or through the column and then to the waste bottle or the
trityl collection port. Functions have been defined to perform each necessary delivery. To achieve
flow of a reagent, specific valves must open simultaneously so that the following occurs:
1. The reservoir is pressurized;
2. The pathway from the reservoir to the valve block is opened; and
3. An exit is provided out of the valve block (i.e. to waste, or to the column and then to waste or
the trityl collection port).
The pathway created by activating a function can be traced using the 391 DNA synthesizer schematic, Figure 4-1. For example, Function 9 (#18 to column) delivers acetonitrile to the column. Activating Function 9 does the following:
1. Opens Valve 17 to allow argon to flow into the acetonitrile reservoir to pressurize it;
2. Opens Valve 1 to open the pathway between the reservoir and the valve block;
3. Opens Valve 12 to open the pathway from the valve block to the column; and
4. Opens Valve 16 to provide an exit (the waste bottle).
Section 4: Functions, Cycles and Procedures
4-5
Applied Biosystems
When all four valves are open, the argon pressure will force the acetonitrile out of the reservoir,
through the reagent valve blocks, through the column and out of the synthesizer to the waste bottle.
When Function 9 is deactivated, all valves close and the flow stops. The pathway created by activating Function 9 is traced using the 391 DNA synthesizer schematic and is shown in Figure 4-2.
Figure 4-2.
4-6
Schematic representing the flow of Function 9 (#18 to column) which opens valves
1, 12, 16 and 17.
Section 4: Functions, Cycles and Procedures
Figure 4-3.
391 FUNCTION LIST
VERSION:
TIME:
DATE:
FUNCTION
NUMBER
1
2
4
5
6
7
9
10
13
14
16
17
19
22
28
31
32
33
34
43
51
52
53
54
55
56
59
60
61
62
63
64
65
66
69
70
71
72
73
74
75
78
81
82
84
85
86
87
88
89
90
1.00
04:27
05/11/89
FUNCTION
NAME
FUNCTION
VALVE LIST
BLOCK FLUSH
REVERSE FLUSH
WAIT
ADVANCE FC
WASTE PORT
WASTE BOTTLE
#18 TO COLM
#18 TO WASTE
#15 TO COLUMN
#14 TO COLUMN
CAP PREP
INTERRUPT
B+TET TO COLUMN
CAP TO COLUMN
PHOS PREP
RCDR ON
RCDR OFF
CYC ENTRY
CYC END
#18 PREP
TET PREP
A TO WASTE
G TO WASTE
C TO WASTE
T TO WASTE
X TO WASTE
CAP A TO WASTE
CAP B TO WASTE
TET TO WASTE
FLUSH TO A
FLUSH TO G
FLUSH TO C
FLUSH TO T
FLUSH TO X
FLUSH TO TET
FLUSH TO #18
#18 TO A
#18 TO G
#18 TO C
#18 TO T
#18 TO X
#18 TO TET
#15 TO WASTE
#14 TO WASTE
#18 TO 14
#18 TO 15
FLUSH TO 14, 15
#18 TO 11
#18 TO 12
FLUSH TO 11, 12
TET TO COLUMN
0, 13, 14, 16
12, 13, 14
Section 4: Functions, Cycles and Procedures
1, 12, 16, 17
1, 13, 17
2, 12, 16, 18
3, 12, 16, 19
22
6, 12, 16, 20, 23
4, 5, 12, 16, 22
20, 23
17
23
11, 13, 20
10, 13, 20
9, 13, 20
8, 13, 20
7, 13, 20
5, 13, 22
4, 13, 22
6, 13, 23
0, 11, 21
0, 10, 21
0, 9, 21
0, 8, 21
0, 7, 21
0, 6
0, 1
1, 11, 17, 21
1, 10, 17, 21
1, 9, 17, 21
1, 8, 17, 21
1, 7, 17, 21
1, 6, 17
2, 13, 18
3, 13, 19
1, 3, 17
1, 2, 17
0, 2, 3
1, 5, 17
1, 4, 17
0, 4, 5
6, 12, 16, 23
4-7
FUNCTION
NUMBER
101
102
103
104
105
106
107
108
109
FUNCTION
NUMBER
92
93
94
95
96
97
98
99
4-8
FUNCTION
NAME
FUNCTION
VALVE LIST
A TO COLUMN
G TO COLUMN
C TO COLUMN
T TO COLUMN
X TO COLUMN
#11 TO COLUMN
#12 TO COLUMN
FLUSH TO TRIT
FLUSH THRU COL
11, 12, 16, 10
10, 12, 16, 20
9, 12, 16, 20
8, 12, 16, 20
7, 12, 16, 20
5, 12, 16, 20
4, 12, 16, 22
0, 12, 15
0, 12, 16
FUNCTION
NAME
FUNCTION
VALVE LIST
USER A
USER B
USER C
USER D
USER E
USER F
USER G
USER H
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Figure 4-4.
MODEL 391 DNA SYNTHESIZER FUNCTIONS
CONTROL
4 - Wait
5 - Advance Fraction Collector
6 - Waste to Port
7 - Waste to Bottle
17 - Interrupt
31 - Recorder On
32 - Recorder Off
33 - Cycle Entry
34 - Cycle End
DELIVER TO COLUMN
9 - #18 to Column
13 - #15 to Column
14 - #14 to Column
19 - Base + Tetrazole to Column
22 - Cap to Column
101 - A to Column
102 - G to Column
103 - C to Column
104 - T to Column
105 - X to Column
90 - TET to Column
106 - #11 to Column
107 - #12 to Column
RINSE AND FLUSH
1 - Block Flush
2 - Reverse Flush
9 - #18 to Column
10 - #18 to Waste
108 - Flush to Trit
109 - Flush thru Col
PREPARE REAGENTS
16 - Cap Prep
28 - Phosphoramidite Prep
43 - #18 Prep
51 - Tetrazole Prep
Section 4: Functions, Cycles and Procedures
PRIME DELIVERY LINES
52 - A to Waste
53 - G to Waste
54 - C to Waste
55 - T to Waste
56 - X to Waste
59 - Cap A to Waste
60 - Cap B to Waste
61 - Tetrazole to Waste
81 - #15 to Waste
82 - #14 to Waste
ARGON TO RESERVOIRS
62 - Flush to A
63 - Flush to G
64 - Flush to C
65 - Flush to T
66 - Flush to X
69 - Flush to Tetrazole
70 - Flush to #18
86 - Flush to 14, 15
89 - Flush to 11, 12
ACETONITRILE TO RESERVOIRS
71 - #18 to A
72 - #18 to G
73 - #18 to C
74 - #18 to T
75 - #18 to X
78 - #18 to Tetrazole
84 - #18 to 14
85 - #18 to 15
87 - #18 to 11
88 - #18 to 12
4-9
Applied Biosystems
Synthesis Cycle Functions
This part of Section 4 categorizes the functions and explains what each function does.
Functions which deliver a reagent to the column
The following five functions provide for flow of a reagent (driven by argon pressure) from its reservoir, through the valve block, through the column and ultimately to the waste bottle. Since the
column is the site of all chemical reactions necessary for synthesis, these deliveries are critical.
FUNCTION
NUMBER
NAME
VALVES
F9
#18 TO COLUMN
1, 17, 12, 16
(Acetonitrile is delivered to the column; frequently used during synthesis to wash the column
and support to remove traces of reagents before a chemical step.)
F 13
#15 TO COLUMN
2, 12, 16, 18
(Iodine is delivered to the column; used to oxidize the DNA following capping)
F 14
#14 TO COLUMN
3, 12, 16, 19
(TCA is delivered to the column; used to detritylate the support-bound oligonucleotides prior
to coupling)
F 19
BASE + TET TO COLM
X, 12, 16, 20, 23, 6
X = any combination
of 7, 8, 9, 10, 11
(According to the sequence, the correct phosphoramidite(s) and tetrazole are |simultaneously
delivered to the column to perform the coupling reaction.)
F 22
CAP TO COLM
4, 5, 12, 16, 22
(Both capping reagents, acetic anhydride (cap A) and NMI (cap B), are simultaneously
delivered to the column; used following coupling to terminate or cap unreacted
oligonucleotide chains.)
F 90
TET TO COLUMN
6, 12, 16, 23
(Tetrazole is delivered to the column; activates phosphoramidites for coupling reaction.)
4-10
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Functions which control external events
The following three functions do not activate solenoid valves, but control electrical switches. They
are used to give electrical signals to a fraction collector or a chart recorder.
FUNCTION
NUMBER
NAME
SWITCH ACTION
F5
ADVANCE FRAC. COLLECT
PULSE
F 31
RECORDER ON
RELAY CLOSE
F 32
RECORDER OFF
RELAY OPEN
Function 5, advance frac. collect (fraction collector), provides a 100-millisecond pulse to Terminals
1 and 2 located on the terminal strip at the left rear of the instrument. When the terminals are connected to a fraction collector, this pulse moves the tube rack one position. The pulse time can be
changed by using the Fract Pulse Menu. The fraction collector is used to collect the trityl cation released during each detritylation step.
Function 31, recorder on, closes an electrical relay or switch and activates an external chart recorder. Function 32, recorder off, and deactivates the chart recorder. The chart recorder should be connected to Terminals 3 and 4 on the rear of the instrument to receive the proper signal. When
connected to a spectrophotometer with a flow cell, a chart recorder can be used to automate trityl
assays. However, these readings will not be quantitative because the trityl solution is too concentrated.
In addition to controlling a chart recorder, these functions can be used to activate and deactivate a
heater. However, be sure not to run any of the heater’s current through the terminals. Contact the
Applied Biosystems Technical Support Department for further information.
Functions which manipulate chemical pathways
The following two functions control the pathway of the column effluent:
FUNCTION
NUMBER
NAME
VALVES
F6
WASTE TO PORT
SUBSTITUTE 15 FOR 16
(Sends the column effluent to the trityl collection port)
F7
WASTE TO BOTTLE
DEFAULT TO 16
(Sends the column effluent to the waste bottle)
Section 4: Functions, Cycles and Procedures
4-11
Applied Biosystems
When Function 6 (waste to port) is activated, all subsequent functions using Valve 16 will have
Valve 15 opened instead. All deliveries to the column will then exit through the trityl collection
port. This is done prior to TCA delivery so that the trityl cation released during detritylation can be
sent to a fraction collector for subsequent analysis.
To return the flow of the column effluent to the waste bottle, Function 7 (waste to bottle) is activated. This signals the controller to open Valve 16 again (default to Valve 16).
Functions which prepare a reagent for delivery
The following functions pressurize reagent reservoirs to prepare them for delivery. A prep (preparation) function opens the gas valve to the reservoir allowing argon to flow to the headspace of the
bottle. After several seconds, the bottle reaches the correct pressure necessary for proper delivery.
These functions are used for reagents which are delivered simultaneously and in equal volumes.
FUNCTION
NUMBER
NAME
VALVES
F 16
CAP PREP
22
(Capping reagents prep (preparation); pressurizes the acetic anhydride and NMI reservoirs
simultaneously.)
F 28
PHOSPHORAMIDITE PREP
20, 23
(Phosphoramidite preparation; pressurizes the tetrazole and all phosphoramidite reservoirs
simultaneously.)
Function 43 and Function 51 are used during the bottle change procedure after fresh tetrazole and
acetonitrile are placed on the instrument. They pressurize the reservoir and blanket it with argon.
F 43
#18 PREP
17
F 51
TET PREP
23
4-12
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Functions used to rinse or flush the chemical pathways
Four functions are frequently used throughout the synthesis to clear the valve blocks, the column
and the interconnecting delivery lines. They are performed prior to a chemical delivery to remove
residual reagent from a previous delivery. Two of the functions, #18 to Column and #18 to Waste,
rinse the pathways with acetonitrile. The other two, Block Flush and Reverse Flush, flush the pathways to remove all liquid. A flush uses argon pressure to force the liquid to the waste bottle.
FUNCTION
NUMBER
NAME
VALVES
F1
BLOCK FLUSH
0, 13, 14, 16
(Removes any solvent or reagent from the reagent valve blocks and the column valve
block; argon enters the three valve blocks simultaneously and forces all liquid to the waste
bottle.)
F2
REVERSE FLUSH
12, 13, 14
(Removes any reagent or solvent from the column and support; argon flows from the
column valve block, through the column, into the reagent valve block and then to waste.
This forces the reagent out of the column in the reverse direction of normal flow (i.e. it
drains the column from the top to the bottom.)
F9
#18 TO COLUMN
1, 17, 12, 16
(Acetonitrile is delivered to the column. This rinses the column and support to remove
traces of reagent. Argon pressure forces the acetonitrile from its reservoir, then through the
reagent valve block, the column, the column valve block and finally to the waste bottle.)
F 10
#18 TO WASTE
1, 17, 13
(Acetonitrile is delivered to the waste bottle. Argon pressure forces acetonitrile from its
reservoir, through the reagent valve blocks to rinse them thoroughly and then to the waste
bottle.)
During synthesis, these four functions are often performed sequentially. For example, function 10
can be activated followed by F 9, F 2 and F 1:
Function 10
Rinses the reagent valve blocks
Function 9
Rinses the column and the column valve block
Function 2
Removes the acetonitrile from the column
Function 1
Removes the acetonitrile from the reagent valve block and the column
valve block
Section 4: Functions, Cycles and Procedures
4-13
Applied Biosystems
Two other functions, 108 and 109, flush the column with argon from the bottom to the top.
F 108
FLUSH TO TRIT
0, 12, 15
(Used following detritylation to flush remaining traces of trityl cation to the trityl collection
port. Produces more accurate trityl assay results.)
F 109
FLUSH THRU COL
0, 12, 16
(Argon flows through the column from the bottom to the top and exits at the waste bottle.)
Functions which prime the reagent valve blocks
Three functions are used to fill or prime the reagent valve blocks prior to column delivery of the
reagent. Argon pressure drives the reagent from its reservoir through the valve block and to the
waste bottle. These functions are also used during the bottle change procedure.
FUNCTION
NUMBER
NAME
VALVES
F 61
TET TO WASTE
6, 13, 23
(Delivers tetrazole to the waste bottle; fills the reagent valve block prior to coupling.)
F 81
15 TO WASTE
2, 13, 18
(Delivers iodine to the waste bottle; fills the reagent valve block prior to oxidation.)
F 82
14 TO WASTE
3, 13, 19
(Delivers TCA to the waste bottle; fills the reagent valve block prior to detritylation.)
Miscellaneous functions
FUNCTION 4
WAIT
Function 4 is a pause which is used following a chemical delivery (such as base + tetrazole to column) to allow the reagents to remain in the column so the reaction can be completed.
FUNCTION 33
CYCLE ENTRY
This function signals the controller to perform the first set of chemical steps necessary for base addition (detritylation). Note that this does not mean the first numerical step of the cycle.
FUNCTION 34
4-14
CYCLE END
Section 4: Functions, Cycles and Procedures
Applied Biosystems
At the end of each cycle, this function signals the controller to perform Step 1 of the cycle which
begins the coupling reaction. Upon completion of all base additions, the cycle will finish at “cycle
entry” if the DNA is to remain trityl on. If the ending method is trityl off, detritylation will be performed and the cycle will finish at “cycle end”.
FUNCTION 17
INTERRUPT
This function is used during the bottle change and shut down procedures. It provides a pause when
reagent bottles are being removed and replaced.
Procedure Functions
The following functions are used during the bottle change, phosphoramidite purge and shut down
procedures. The bottle change procedure is used to remove empty reservoirs and replace them with
fresh reagents. The phosphoramidite purge procedure fills the phosphoramidite and tetrazole delivery lines with fresh reagent prior to beginning a synthesis. The shut down procedure prepares the
instrument for storage. It removes all reagents from the delivery lines, and washes and dries all
chemical pathways.
Functions which prime delivery lines
When a bottle of fresh reagent is placed on the instrument, the delivery line from the reservoir to
the valve block must be primed or filled with the reagent. To prime the line, argon pressure forces
the chemical from its reservoir, through the reagent valve blocks and to the waste bottle.
Functions 52-56 and 61 are also used during the phosphoramidite purge procedure. Prior to synthesis, these functions fill the phosphoramidite and tetrazole lines with fresh reagent. In addition, Functions 61, 81 and 82 are used during the synthesis cycle to fill the reagent valve block prior to column
delivery.
The following functions deliver the contents of the designated reservoir to the waste bottle. For example, Function 52, A to waste, delivers adenosine phosphoramidite to the waste bottle.
Section 4: Functions, Cycles and Procedures
4-15
Applied Biosystems
FUNCTION
NUMBER
NAME
VALVES
F 52
F 53
F 54
F 55
F 56
F 59
A TO WASTE
G TO WASTE
C TO WASTE
T TO WASTE
X TO WASTE
CAP A TO WASTE
(acetic anhydride to waste)
CAP B TO WASTE
(NMI to waste)
TET TO WASTE
#15 TO WASTE
#14 TO WASTE
11, 13, 20
10, 13, 20
9, 13, 20
8, 13, 20
7, 13, 20
5, 13, 22
F 60
F 61
F 81
F 82
4, 13, 22
6, 13, 23
2, 13, 18
3, 13, 19
Functions which deliver acetonitrile to a reservoir
Before removing an empty phosphoramidite or tetrazole bottle, the old reagent in the delivery line
is forced back into the reservoir and the line is rinsed. This is done during the bottle change procedure by delivering acetonitrile through the reagent valve block to the empty reservoir. In addition,
functions delivering acetonitrile to bottle positions 11, 12, 14 and 15 are used during the shut down
and flow test procedures to remove the reagents from the delivery lines.
The following functions deliver acetonitrile to the designated reservoir. For example, Function 71,
#18 to A, delivers acetonitrile to the adenosine phosphoramidite reservoir.
FUNCTION
NUMBER
NAME
VALVES
F 71
F 72
F 73
F 74
F 75
F 78
F 84
F 85
F 87
F 88
#18 TO A
#18 TO G
#18 TO C
#18 TO T
#18 TO X
#18 TO TET
#18 TO 14
#18 TO 15
#18 TO 11
#18 TO 12
1, 11, 17, 21
1, 10, 17, 21
1, 9, 17, 21
1, 8, 17, 21
1, 7, 17, 21
1, 6, 17
1. 3. 17
1, 2, 17
1, 5, 17
1, 4, 17
4-16
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Functions which deliver argon to a reservoir
These functions are used during the bottle change procedure for the phosphoramidite, tetrazole, and
acetonitrile reservoirs. After acetonitrile removes old reagent from the delivery line, the acetonitrile
is removed by an argon flush. Argon travels through the reagent valve block (by opening Valve 0)
and into the reservoir which forces the acetonitrile into the bottle. The phosphoramidite bottles
share a single-vent valve which is open during this step. In addition, functions that deliver argon to
bottle positions 11, 12, 14 and 15 are used during the shut down and flow test procedures.
The functions below deliver argon to the designated reservoir. For example, Function 62, Flush to
A, delivers argon to the adenosine phosphoramidite reservoir.
FUNCTION
NUMBER
NAME
VALVES
F 62
FLUSH TO A
0, 11, 21
F 63
FLUSH TO G
0, 10, 21
F 64
FLUSH TO C
0, 9, 21
F 65
FLUSH TO T
0, 8, 21
F 66
FLUSH TO X
0, 7, 21
F 69
FLUSH TO TET
0, 6
F 70
FLUSH TO #18
0, 1
F 86
FLUSH TO 14, 15
0, 2, 3
F 89
FLUSH TO 11, 12
0, 4, 5
Section 4: Functions, Cycles and Procedures
4-17
Applied Biosystems
Test Functions
Test functions are used to perform the flow test procedure.The procedure is used during instrument
manufacturing, installation and troubleshooting. If desired, test functions can be inserted into a synthesis cycle using the Cycle Editor or a procedure using the Procedure Editor. The following functions deliver the contents of the designated bottle to the column.
FUNCTION
NUMBER
NAME
VALVES
F 101
A TO COLUMN
11, 12, 16, 20
F 102
G TO COLUMN
10, 12, 16, 20
F 103
C TO COLUMN
9, 12, 16, 20
F 104
T TO COLUMN
8, 12, 16, 20
F 105
X TO COLUMN
7, 12, 16, 20
F 90
TET TO COLUMN
6, 12, 16, 23
F 106
#11 TO COLUMN
5, 12, 16, 22
F 107
#12 TO COLUMN
4, 12, 16, 22
(Note that when you print the functions, F108 and F109 are listed with the test functions. When categorizing, they are included with functions that rinse and flush chemical pathways.)
Synthesis Cycles
A function performed for a specified time is called a step. During synthesis, steps are arranged in a
particular order to create a synthesis cycle. The cycle completes all operations necessary for one
base addition and is repeated until DNA of desired length is fully synthesized.
Applied Biosystems supplies four synthesis cycles which are optimized for use with β-cyanoethyl
phosphoramidites. They are stored permanently in the ROM cycle locations of the Cycle Editor.
They cannot be changed or deleted. The cycles are used for synthesizing on the ten-micromole, onemicromole and small scales (0.2 micromole) and are named “10µM”, “1µM” and “.2µM” respectively. A low reagent consumption cycle on the 0.2µM scale has also been developed which uses
33% less phosphoramidites. This cycle is called “Low”.
When the instrument’s main power is first turned on, all ROM cycles (.2µM, IµM, Low, 10µM) are
automatically loaded into the four RAM cycle locations of the Cycle Editor. See Table 4-1 for the
exact locations. Once in a RAM location, any cycle can be edited and all changes will be stored in
memory even if the power is turned off. In the Start Synthesis Menu, you must choose a RAM cycle
4-18
Section 4: Functions, Cycles and Procedures
Applied Biosystems
for use in DNA synthesis, the ROM cycles do not appear as options. Note that at any time, you can
copy a ROM cycle into a RAM cycle location by using the Cycle Editor. You can also create entirely new cycles in any RAM location.
ROM
synthesis
cycle
RAM
cycle
location
Total
Number
of steps
cycle
time
(min.)
crude
yield* (O.D.)
(20mer)
.2µM
Cycle-1
63
5.5
20-25
1µM
Cycle-2
64
5.5
100-120
Low**
Cycle-3
61
5.5
20-25
10µM
Cycle-4
53
25
800-1000
*Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming 33 micrograms/O.D. unit.
**Low is a low consumption cycle on the .2µM scale.
Table 4-1. Applied Biosystems Synthesis Cycles
The 0.2-µMol scale provides sufficient oligomers for most applications. Using this scale, the expected crude yield in optical density units (O.D. or odu, A260 nm.) equals the number of bases in
the sequence. For example, synthesis of a 20-mer typically yields approximately 20 O.D. of crude
oligonucleotide. When larger quantities of DNA are needed, the 1µM or l0µM cycle can be used.
With the 1µM cycle, synthesis of a 20-mer will yield about 100 O.D. of crude oligonucleotide (1µM
crude yield, O.D. = length x 5). Synthesizing a 20-mer with the l0µM cycle will yield about 800 odu
of crude DNA.
The steps in each cycle have optimized delivery times which ensure completion of each chemical
reaction, yet an entire synthesis cycle is complete in 5.5 minutes. (The cycle time for l0µM is 25
minutes.) The 0.2µM cycle cost is about 45% less than the 1µM cycle cost. The “Low” cycle costs
are about 25% less than the 0.2µM cost.
All cycles are listed in Appendix A. In addition, the .2µM cycle is shown in Figure 4-5. Each cycle
consists of the required number of steps which are listed with the corresponding function number,
name and step time. To determine the purpose of each step, refer to the explanation of Synthesis
Cycle Functions found earlier in this section.
Section 4: Functions, Cycles and Procedures
4-19
Applied Biosystems
More About the .2µM Cycle
The four essential chemical reactions necessary for synthesis are listed below with their corresponding cycle step numbers:
1.
2.
3.
4.
Detritylation
Coupling
Capping
Oxidation
.2µM
Steps 36-63
Steps 1-15
Steps 16-21
Steps 22-35
Although each reaction requires different treatment, the following generalizations can be made:
Prior to the chemical reaction:
• The valve blocks, the column and the interconnecting delivery lines are rinsed and flushed
dry; and
• The reagent reservoir(s) is prepared for delivery.
To perform the chemical reaction:
• The reagent(s) is delivered to the column, often followed by a Wait step to complete the
reaction.
4-20
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Figure 4-5.
STEP
NUMBER
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
.2µΜ β−cyanoethyl synthesis cycle
FUNCTION
#
NAME
10
9
2
1
28
90
19
90
19
90
19
90
4
16
2
1
22
4
10
2
1
81
13
10
4
2
1
10
9
2
9
2
9
2
1
33
10
9
2
1
5
6
82
14
108
14
#18 TO WASTE
# 18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
PHOS PREP
TET TO COLUMN
B+TET TO COLM
TET TO COLUMN
B+TET TO COLM
TET TO COLUMN
B+TET TO COLM
TET TO COLUMN
WAIT
CAP PREP
REVERSE FLUSH
BLOCK FLUSH
CAP TO COLUMN
WAIT
#18 TO WASTE
REVERSE FLUSH
BLOCK FLUSH
#15 TO WASTE
#15 TO COLUMN
#18 TO WASTE
WAIT
REVERSE FLUSH
BLOCK FLUSH
#18 TO WASTE
#18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
CYC ENTRY
#18 TO WASTE
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
ADVANCE FC
WASTE PORT
#14 TO WASTE
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
Section 4: Functions, Cycles and Procedures
STEP
TIME
2
15
5
3
3
3
2
2
2
2
2
2
15
3
5
3
10
5
3
5
3
3
10
5
15
5
3
5
10
5
10
5
10
5
3
1
3
10
5
3
1
1
3
10
1
10
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
(continued →)
4-21
Applied Biosystems
Figure 4-5.
STEP
NUMBER
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
4-22
.2µΜ β−cyanoethyl synthesis cycle (Continued)
FUNCTION
#
NAME
108
14
108
14
108
14
108
14
108
9
108
7
1
9
2
1
34
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#18 TO COLM
FLUSH TO TRIT
WASTE BOTTLE
BLOCK FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
CYC END
STEP
TIME
1
10
1
10
1
10
1
10
1
10
8
1
3
20
5
3
1
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Section 4: Functions, Cycles and Procedures
Applied Biosystems
Procedures
The Phosphoramidite Purge Procedure
The phosphoramidite purge procedure fills all phosphoramidite and tetrazole delivery lines (from
the reservoir to the reagent valve block) with fresh reagent. This process uses less than one-half the
amount of phosphoramidite used in a coupling step of a one-micromole synthesis. A purge is done
prior to beginning a synthesis when the instrument has been idle for more than 12 hours (more than
6 hours in humid environments) or if one of the phosphoramidite reservoirs has not been accessed
within 12 hours. Over this time, oxygen and atmospheric water can penetrate the delivery lines and
cause failures in the first coupling. A phosphoramidite purge eliminates this problem and helps ensure a successful synthesis.
IMPORTANT Not performing a phosphoramidite purge when it is needed will cause a synthesis to
fail. When in doubt, it is best to purge since it may save a synthesis.
Before beginning a synthesis, you must choose whether to perform the procedure. Refer to Section
3: Main Menu Option: Start Synthesis for further information. Note that a phosphoramidite purge
is not included in the bottle cycle counter which is used to tabulate the alarm.
As shown below, the procedure consists of nine steps. If desired, the steps can be changed using the
Procedure Editor Menu.
The Phosphoramidite Purge Procedure
STEP
NUMBER
1
2
3
4
5
6
7
FUNCTION
NUMBER
FUNCTION
NAME
TIME
SECONDS
F 28
PHOS PREP
10
(pressurizes the phosphoramidite and tetrazole reservoirs prior to delivery)
F 52
A TO WASTE
4
F 53
G TO WASTE
4
F 54
C TO WASTE
4
F 55
T TO WASTE
4
F 56
X TO WASTE
4
F 61
TET TO WASTE
4
(Steps 2-7 deliver the contents of the designated reservoir to the waste bottle. Fills the
delivery lines and the reagent valve block)
Section 4: Functions, Cycles and Procedures
4-23
Applied Biosystems
STEP
NUMBER
8
9
FUNCTION
NUMBER
FUNCTION
NAME
TIME
SECONDS
F 10
#18 TO WASTE
7
(Removes the phosphoramidite and tetrazole left in the reagent valve block. Rinses the valve
block)
F1
BLOCK FLUSH
10
(Removes acetonitrile from the reagent valve blocks)
The Bottle Change Procedure
Bottle change procedures are used to remove empty reservoirs and replace them with bottles of
fresh reagents. They can be performed before beginning a synthesis or when a synthesis is interrupted either manually or by the alarm. This process is especially important for preventing oxygen and
water contamination of atmosphere-sensitive phosphoramidites and tetrazole. A procedure exists
for all 11 bottles and each can be edited using the Procedure Editor. Copies of the procedures can
be found in Appendix A.
4-24
Section 4: Functions, Cycles and Procedures
Applied Biosystems
The Phosphoramidite Bottle Change Procedure
STEP
NUMBER
FUNCTION
NUMBER
FUNCTION
DESCRIPTION
TIME
SECONDS
1
2
F1
BLOCK FLUSH
5
F 10
18 TO WASTE
7
(The reagent valve blocks are cleared by F1, block flush, for 5 seconds and rinsed by F 10,
#18 to waste, for 7 seconds.)
3
A-PHOS, F 71
18 TO BOTTLE
5
G-PHOS, F 72
C-PHOS, F 73
T-PHOS, F 74
X-PHOS, F 75
(Old reagent in the delivery line is removed and forced back into the reservoir by an
acetonitrile wash via delivering #18 to the designated bottle for 5 seconds.)
4
A-PHOS, F 62
FLUSH TO BOTTLE
10
G-PHOS, F 63
C-PHOS, F 64
T-PHOS, F 65
X-PHOS, F 66
(An argon flush clears the lines for 10 seconds)
5
INTERRUPT, F 17
(Remove the bottle, place the new bottle on the instrument)
If you are going to remove the phosphoramidites and save them for later use, do not complete these
steps or HPLC grade acetonitrile will contaminate the bottles. Refer to Section 2 for instructions
on how to store dissolved phosphoramidites.
6
A-PHOS, F 62
FLUSH TO BOTTLE
5
G-PHOS, F 63
C-PHOS, F 64
T-PHOS, F 65
X-PHOS, F 66
(Argon purges the headspace of the phosphoramidite reservoirs to eliminate air.)
7
A-PHOS, F 52
BOTTLE TO WASTE
4
G-PHOS, F 53
C-PHOS, F 54
T-PHOS, F 55
X-PHOS, F 56
TETRAZOLE, F 61
(The delivery line is refilled (primed) with fresh reagent by delivering the contents of the
designated bottle to waste for 10 seconds.)
8
F 10
18 TO WASTE
7
9
F1
BLOCK FLUSH
10
(The reagent valve blocks are rinsed by F 10, #18 to waste, for 7 seconds and cleared by
F 1, block flush, for 10 seconds.)
Section 4: Functions, Cycles and Procedures
4-25
Applied Biosystems
Similarly, the bottle change for acetonitrile (Bottle 18) begins by performing F 1, block flush, for 5
seconds and F 10, #18 to waste, for 7 seconds. The bottle is then removed and replaced. Finally,
fresh acetonitrile is delivered to waste (F 10 for 7 seconds) and the reagent valve block is cleared
by a block flush, F 1, for 10 seconds.
Both capping reagents (cap A, acetic anhydride, Bottle 11; cap B, NMI, Bottle 12) use the same
procedure. The valve blocks are cleared and rinsed by F 1 for 5 seconds and F 10 for 7 seconds. The
bottles are then removed and replaced. Next, argon pressurizes the reservoir by F 16, cap prep, for
5 seconds. Then, the contents of the bottles are delivered to waste to prime the lines (using F 59 for
acetic anhydride and F 60 for NMI for 5 seconds). Finally, the valve blocks are rinsed and cleared
by F 10 for 7 seconds, and F 1 for 10 seconds.
The TCA (Bottle 14) and iodine (Bottle 15) reservoirs use the same bottle change procedure. The
valve blocks are cleared and rinsed by F 1 for 5 seconds and F 10 for 7 seconds. The bottles are then
removed and replaced. Next, the delivery lines are primed for 5 seconds (F 82 for TCA; F 81 for
iodine). Finally, the valve blocks are rinsed and cleared by F 10 for 7 seconds and then F 1 for 10
seconds.
4-26
Section 4: Functions, Cycles and Procedures
Applied Biosystems
The Shut Down Procedure
The shut down procedure prepares the instrument for long term storage. It removes all reagents in
the delivery lines and washes and dries all chemical pathways. To perform this procedure, select
Shut Down from the main menu and follow the instructions in Section 3.
STEP
NUMBER
FUNCTION NUMBER
FUNCTION
DESCRIPTION
TIME
SECONDS
1
2
3
4
5
6
7
F1
BLOCK FLUSH
5
F 10
18 TO WASTE
5
F 71
#18 TO A
60
F 72
#18 TO G
60
F 73
#18 TO C
60
F 74
#18 TO T
60
F 75
#18 TO X
60
(Old phosphoramidites in the delivery lines are removed and forced back into the reservoirs
by an acetonitrile wash.)
8
F 62
FLUSH TO A
60
9
F 63
FLUSH TO G
60
10
F 64
FLUSH TO C
60
11
F 65
FLUSH TO T
60
12
F 66
FLUSH TO X
60
(The lines are cleared by an argon flush to each bottle)
13
10
#18 TO WASTE
10
14
78
#18 TO TET
60
15
87
#18 TO 11
60
16
88
#18 TO 12
60
17
84
#18 TO 14
60
18
85
#18 TO 15
60
(Reagents in these delivery lines are removed and forced back into the reservoirs by an
acetonitrile wash)
19
69
FLUSH TO TET
60
20
89
FLUSH TO 11, 12
60
21
86
FLUSH TO 14, 15
60
(The lines are cleared by an argon flush to each bottle)
22
10
18 TO WASTE
10
23
1
BLOCK FLUSH
5
24
17
INTERRUPT
1
(F 17 Provides a pause to remove all bottles and wipe the lines)
25
62
FLUSH TO A
5
26
63
FLUSH TO G
5
27
64
FLUSH TO C
5
28
65
FLUSH TO T
5
29
66
FLUSH TO X
5
30
70
FLUSH TO 18
10
(Provides an additional argon flush to clear the phosphoramidite and acetonitrile lines)
31
1
BLOCK FLUSH
10
Section 4: Functions, Cycles and Procedures
4-27
Applied Biosystems
The Flow Test Procedure
The flow test procedure is used to measure flow rates through all essential delivery lines. It can be
used for routine maintenance, troubleshooting and recalibration of the instrument if you suspect
flow related problems.
To perform this procedure, select SELF TEST from the Main Menu. Then, press MORE until the
FLOWTST option is shown. Further instructions appear in Section 3: Main Menu Option: Self Test
and Section 2: How to Perform the Flow Test Procedure.
This procedure is not found in the procedure editor and cannot be edited or printed. A copy of the
flow test procedure can be found in Appendix A.
The flow test has four parts and takes about one hour to perform. The first part, steps 1-14, washes
and primes the delivery lines with acetonitrile. The second, steps 15- 43, measures the flow of acetonitrile from each bottle position to the lower column Luer fitting. The third part, steps 44-46, measures the flow from bottle 14 to the trityl collection line. In the final part, steps 47-59, all the lines
are flushed dry with argon enabling reattachment of the reagents.
4-28
Section 4: Functions, Cycles and Procedures
Section 5:
System Description – Hardware
This section describes the components of the Model 391 DNA Synthesizer. Use it as a reference
guide to familiarize yourself with the synthesizer and its operations.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-2
The Chemical Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-3
Pressure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4
Reagent and Solvent Reservoirs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4
Delivery Valve Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-6
Vacuum Assist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-9
The Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-10
Waste and Venting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-11
The Battery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-12
The Controller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-13
Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-14
Applied Biosystems
Introduction
The major components of the Model 391 Automated DNA Synthesizer are shown in Figure 5-1.
All chemical steps required for DNA synthesis take place within a reaction chamber, called the column. The column contains the 3'-terminal nucleoside which is covalently attached to a support. The
DNA chain is built by adding one base at a time to the support-bound nucleoside.
All reagents and solvents necessary for synthesis are accessed by the chemical delivery system. In
this system, a set of solenoid valves opens to create a pathway for chemical flow. Pressure regulated
argon forces the chemicals to flow from their reservoirs through the pathway (consisting of one or
more valve blocks and delivery lines) and then to the column. After completing a chemical step, the
column effluent flows to either the waste bottle, or the trityl collection port to collect the dimethoxytrityl cation for analysis.
The controller directs and initiates all synthesizer activity and consists of a microprocessor, the software, a keyboard, a display screen and associated electronics. Information is displayed on a twoline screen and you give instructions by pressing the appropriate key on the keyboard.
5-2
Section 5: System Description – Hardware
Applied Biosystems
The Chemical Delivery System
The flow of reagents, solvents and gas through the Model 391 is controlled by a positive-pressure
chemical delivery system. The system components include a regulated pressure source, eleven reagent and solvent reservoirs, delivery valves, a column, a vented waste bottle and delivery lines
which interconnect the components. All inner surfaces of the chemical delivery system are made of
inert materials.
Figure 5-1.
Major Components of the Model 391
Section 5: System Description – Hardware
5-3
Applied Biosystems
Pressure
System pressure is provided by pre-purified (99.998%) argon. Argon’s high density and low oxygen
contamination make it preferable to nitrogen. An argon cylinder is connected to the inlet port at the
right rear of the Model 391 using ¼-inch oxygen-impermeable tubing and a gas-tight connector supplied by Applied Biosystems. The pressure regulator on the tank is set to 60 psi. This high pressure
argon is used to operate the vacuum-assist system (described later in this section) and to supply argon to a regulator in the synthesizer.
IMPORTANT The argon cylinder must always be connected to the synthesizer before the power is
turned on. If this is not done, the vacuum-assist valves will be activated continuously
and can overheat and fail causing argon to leak.
An empty argon cylinder can be replaced before beginning a synthesis or when an active synthesis
is interrupted. Refer to Section 2: How to Change an Argon Tank.
Argon entering the synthesizer travels through a 10-micron particle filter to a pressure regulator.
The regulator delivers argon to specific pressure valves used to pressurize the reagent and solvent
reservoirs. (Pressure valves are further discussed in Section 4: Valves, and are shown in Figure 41.) In addition, the regulator supplies argon to the column valve block and the reagent valve blocks.
The phosphoramidites have one pressure valve which supplies argon to a manifold pressurizing all
five phosphoramidite bottles simultaneously. For proper pressurization, bottles must be attached to
all five positions, even if some are empty. The two capping reagents (1-methylimidazole, NMI, and
acetic anhydride) share a pressure valve which channels the argon to a tee to pressurize both bottles.
All other reagent reservoirs (tetrazole, trichloroacetic acid, iodine and acetonitrile) are pressurized
using a single valve for each bottle.
The regulator is adjusted so that acetonitrile from bottle 18 has a flow rate of 2.80 to 3.20 mL/min
measured at the lower Luer fitting of the column. The pressure gauge should read approximately
4.0 psi. For details on how to adjust flow rates, refer to Section 2: How to Perform the Flow Test
Procedure.
Reagent and Solvent Reservoirs
Each reservoir has a unique position on the instrument and is referred to by the number located
above each receptacle. Position numbers are also printed on the bottle labels. The position or reservoir numbers are 1-5, 9, 11, 12, 14, 15, and 18. These numbers correspond to those on other Applied
Biosystems DNA synthesizers which have 18 positions.
Reservoirs 1-5 are pushed upward around a Teflon insert and a silicone O-ring to form an airtight
seal inside each bottle neck. Pressing the appropriate black button above the bottle position releases
5-4
Section 5: System Description – Hardware
Applied Biosystems
the grip on the bottle and allows for its removal and subsequent replacement. When replacing a bottle, the button will return to its out position only when the reservoir is correctly engaged.
Bottles 9, 11, 12, 14 and 15 screw snugly into a threaded cap mounted on the synthesizer. A disposable polyethylene insert forms an airtight seal between each cap and reservoir.
Note
Receptacles 9-15 have a ratchet cap assembly. When attaching these bottles, you
cannot overtighten them.
Reservoir 18, containing acetonitrile, is a 4-Liter bottle that does not attach directly to the instrument. Instead, you place it inside a protective carrier and put it into a metal rack that attaches to the
left side of the instrument. A cap assembly, which includes the delivery and gas lines, screws onto
the bottle and connects to the synthesizer. The cap has a teflon insert and a silicone rubber gasket.
Applied Biosystems provides the cap assembly, the carrier, the rack and one bottle of acetonitrile.
The Pressure and Delivery Lines
Each bottle has an argon pressure line and a delivery line entering through the cap insert. For the
phosphoramidites, bottles 1-5, the pressure line also functions as a vent line. As shown in Figure 52, the argon line remains above the liquid level while the delivery line extends to the bottom of the
bottle. Upon opening the correct set of valves, the reservoir headspace is pressurized by argon and
the liquid is pushed into the delivery line and flows to its destination.
Figure 5-2.
Reagent Reservoir
Since all phosphoramidite reservoirs are pressurized simultaneously by a single valve, bottles must
be attached to all 5 positions (even if some are empty) or the argon will escape out any exposed
pressure line. Also, bottles should be placed on all positions to keep the lines clean.
Notice that all lines are color coded. Phosphoramidite delivery lines are red (0.5mm ID), while the
other reagent delivery lines are blue (0.8mm ID). All pressure lines are blue (0.8mm ID).
Section 5: System Description – Hardware
5-5
Applied Biosystems
Venting
In addition to being pressurized, the phosphoramidites are vented using the pressure/vent line.
When fresh phosphoramidites, which are atmosphere-sensitive, are placed on the instrument, they
are purged with argon to eliminate air. A purge delivers gas through the delivery line. As the gas is
passed through the bottle, the air escapes out the pressure/vent line. This is performed automatically
by the bottle change procedure.
Delivery Valve Blocks
The chemical delivery system includes two reagent valve blocks and one column valve block.The
valve blocks control gas and chemical flows to the column and exit ports. Figure 5-3 shows a schematic representation of all three valve blocks.
5-6
Section 5: System Description – Hardware
Applied Biosystems
Figure 5-3.
Valve Blocks Schematic
Section 5: System Description – Hardware
5-7
Applied Biosystems
The first reagent valve block is an eight-port block which controls the delivery of the following solvents and reagents:
tetrazole
acetic anhydride
1-methylimidazole
trichloroacetic acid
iodine
acetonitrile
bottle 9
bottle 11
bottle 12
bottle 14
bottle 15
bottle 18
Each of the above reagents flows from its reservoir through the first reagent valve block and then
through the second which directs the flow to the column or waste. The first reagent valve block also
controls the delivery of argon through both reagent valve blocks.
The second reagent valve block, an eight-port block, controls the delivery of the phosphoramidites
to the column or waste. It also directs the flow from the first reagent valve block to the column or
waste.
The column valve block, a four-port block, directs the column effluent to either the waste or the
trityl collection port. It also controls the argon gas used to remove or flush the reagents from the
column and the column valve block.
The design of the valve blocks provides zero-dead volume. Delivery lines feed into each valve
block and connect to the common pathway in the valve block manifold via a manifold inlet line and
a solenoid-controlled diaphragm valve. See Figure 5-4.
The delivery lines enter the valve block through the manifold inlet lines. Passage between the manifold inlet line and the common pathway of the valve block is accomplished by an open solenoid
valve. When a valve opens, the solenoid piston pulls away from a diaphragm located under the piston. With vacuum assist, an open solenoid will cause the diaphragm to form a 2-µL domed chamber.
The domed chamber creates a passageway between the inlet line and the common pathway. The
common pathway zigzags through the valve block manifold and passes other closed valves which
are unaffected by the flow. The direction of flow is determined by the pressures on either side of
the valve block. During proper operation, the flow will be toward the column or one of the exit
ports.
5-8
Section 5: System Description – Hardware
Applied Biosystems
Figure 5-4.
The Valve Block
Vacuum Assist
For proper valve block operation, it is crucial that each diaphragm forms a domed chamber. The
vacuum assist creates a vacuum on the solenoid side of the diaphragm to form a domed chamber
each time a solenoid valve opens. An aspirator pump provides vacuum assist to each valve block.
The aspirator pump requires that regulated argon enter the synthesizer at a gauge pressure of approximately 60 psi. The argon regulator should be connected to the instrument with the ¼-inch tubing supplied by Applied Biosystems.
IMPORTANT Do not use 1/8-inch tubing, it is too narrow to deliver the correct volume of gas.
The vacuum gauge should always read between 14 in. and 18 in. Hg. When the gauge drops below
14 in. Hg. (once every 4-10 hours), a valve opens to allow high pressure argon to flow to the aspirator pump. The gas is then diverted out of the pump and creates a Venturi vacuum when a second
Section 5: System Description – Hardware
5-9
Applied Biosystems
valve opens to the vacuum-assist plumbing. If the gauge pressure drops below 14 in. Hg. more than
once every 4 hours, there is a leak in the system. Contact the Applied Biosystems Technical Support
Department for repair instructions.
The Column
The initial support-bound nucleoside is contained in a disposable column which has two silica-retaining filters, two end fittings and two caps (Figure 5-5). All parts, except the aluminum caps, are
made of inert materials. The retaining filters are porous Teflon held in place by a circular ridge on
the inside of the column. Under pressure from the metal cap, the end fittings press against the ridge
to form a tight seal around the edge of each filter. Tear-off aluminum tabs protect the filters during
shipping. The inlet and outlet are female Luer fittings designed to mate with the male Luer fittings
on the instrument. The column is symmetrical (i.e. no top or bottom, no front or back) and can be
attached to the male Luer fittings in any way. Each column is color coded to show the initial nucleoside and has a unique serial number.
Column
A
G
C
T
Color code
green
yellow
red
blue
The normal flow into the column is from the bottom. By sending the liquid stream upward, the CPG
particles are lifted and maintained in a fluidized state. The flow rates of the solvents and reagents
have been set to achieve proper mixing of the particles.
5-10
Section 5: System Description – Hardware
Applied Biosystems
Figure 5-5.
The Column
Waste and Venting
The ultimate destination of most chemical deliveries is the waste bottle. The bottle is a free-standing, four-liter polyethylene container which should be placed on the floor near the synthesizer or on
a nearby bench lower than the instrument. The bottle can be kept inside a protective bottle carrier
to contain accidental spillage. A one-gallon carrier is sufficient and can be purchased from VWR,
Part Number 56609-186, or Nalge, Part Number 6501-0010.
Section 5: System Description – Hardware
5-11
Applied Biosystems
WARNING
Synthesizer waste must be handled and disposed of properly and carefully.
Depending on the synthesis cycle used, the instrument generates 1 to 2 liters
of hazardous, halogenated organic liquid waste per 100 cycles of operation.
When handling the waste for disposal, wear gloves, a lab coat and eye
protection, and avoid inhalation and skin contact. Place the liquid in a sealed
container labeled “FLAMMABLE”, “POISON B.N.O.S.” or absorb in vermiculite,
dry sand or earth. Dispose of properly according to the appropriate local
government regulations.
A waste line and vent line enter the cap of the waste bottle. Be sure this cap is always securely tightened. The waste line carries liquid waste from the synthesizer to the bottle. Be sure the line slopes
downward toward the bottle and has no troughs that can collect waste and block the line.
The vent line carries gaseous waste to a suitable exhaust such as a fume hood. Applied Biosystems
supplies a 25-foot length of tubing to connect the vent line to a fume hood. Proper venting allows
operation of the Model 391 on an open lab bench.
IMPORTANT Prevent condensation from collecting in the vent line by continuously sloping the
tubing upward toward the fume hood.
The tubing should not be horizontal or have troughs that will form collecting points. Condensation
will eventually cause erratic deliveries and can even stop the flow.
IMPORTANT The waste bottle is the low pressure side of the delivery system and must always be
vented to atmosphere. If it is not, back pressure will be generated which will decrease
the deliveries of reagents and solvents.
The Battery
A lithium battery is provided in the Model 391. When the main power is turned off (either intentionally or during a power failure), all synthesis parameters are retained. These parameters include
the DNA sequences; user-defined cycles, procedures and functions; and bottle usage information.
If a power failure occurs during an active synthesis, the synthesis will be interrupted and can only
be resumed when the main power returns.
The battery also maintains the synthesizer’s internal clock. If a power outage does occur, the clock
records the time the power failed and the time it was restored. A synthesis will then automatically
resume if the elapsed time of the failure is less than the time you entered in the Power Fail Menu.
Refer to Section 3: Main Menu Option: Power Fail, for further instructions.
5-12
Section 5: System Description – Hardware
Applied Biosystems
The battery’s voltage is checked during the system self-test of the electronic components. It should
operate for several years. When it needs replacing, the display screen will show a battery low message.
WARNING
The used lithium battery must be replaced and disposed of properly. For
further information, consult the Applied Biosystems Technical Support
Department.
The Controller
The controller directs and initiates all synthesizer activity. Its major components are the software,
the microprocessor, the display screen, the keyboard and associated electronics.
The software defines all necessary operations for synthesis and is interpreted and executed by the
microprocessor. The software is contained within a removable memory cartridge which plugs into
the rear of the instrument. Updated software supplied by Applied Biosystems can be easily installed
by removing the old cartridge and replacing it with the new one.
IMPORTANT Before removing the memory cartridge, be sure the main power is off.
Synthesis information is shown on a two-line liquid crystal display (LCD). The display can be adjusted for optimal viewing by turning the knob at the left of the screen.
You interact with the instrument by selecting keys on the keyboard. The keyboard consists of five
soft keys (located below the screen) and several labeled keys (0-9; A, G, C, T, X; delete; and left
and right arrows). The labels and actions of the soft keys change depending on the information displayed directly above them.
The software is “menu-driven” where menus and pages of menus present various options and necessary information about the synthesis or status of the instrument. In response, you select an option
and give instructions by pressing the appropriate key. For a complete description of all menus, refer
to Section 3.
To perform automated synthesis, the software uses a cycle consisting of a series of steps which complete all chemical reactions. For further information about cycles, see Section 3: Main Menu Option: Cycle Editor, and Section 4: Synthesis Cycles.
Proper operation of the controllers electronic components are verified by a system self test. For further instructions, refer to Section 3: Main Menu Option: Self Test.
Section 5: System Description – Hardware
5-13
Applied Biosystems
IMPORTANT A fan on the rear of the instrument distributes air through the electronics for cooling
and should not be obstructed.
Fuses
The Model 391 has two power fuses. They are located at the rear of the instrument to the left of the
fan and directly above the power line. Although the fuses have a very long life, they can stop operating. For example, if the 391 has no power (i.e. the fan is not working or the LCD screen is blank),
a bad fuse may be causing the problem and will need replacing. Before concluding there is a bad
fuse, however, check if the instrument is properly connected to a functional power source. Extra
fuses are included in the spare parts kit. For further information call the Applied Biosystems Technical Support Department.
5-14
Section 5: System Description – Hardware
Section 6:
Chemistry for Automated DNA
Synthesis
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-2
The Solid Support - CPG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5
DNA Synthesis Chemistry Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-7
Detritylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-8
Monitoring the trityl cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-9
Depurination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-11
Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-12
Phosphoramidites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-12
Capping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15
Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-16
Completion of the Synthesis Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18
Manual Deprotection and Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18
Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18
Phosphate Deprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18
Base Deprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-19
Quantitation of the Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-20
Storage of the Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-20
Analysis and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-21
OPC, Oligonucleotide Purification Cartridge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-21
PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-22
HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24
Alternative Chemistries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24
RNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24
Hydrogen-Phosphonate Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-25
Phosphorothioate DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-26
5' Attachments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-27
Fluorescent-dye Linked Sequencing Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-28
Amino Link 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-28
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-30
Applied Biosystems
Introduction
DNA synthesis is quite simple in concept. A reactive 3' phosphorous group of one nucleoside is coupled to the 5' hydroxyl of another nucleoside. The former is a monomer, delivered in solution. The
latter is immobilized on a solid support. An internucleotide linkage is thus formed. Three other
chemical reactions are necessary to prepare the growing chain of DNA for the next coupling. In this
way a synthesis cycle is conducted, adding one nucleoside monomer at a time. The desired sequence
and length are defined by the operator on the synthesizer.1 When the chain is complete, the crude
DNA (oligonucleotide) must be cleaved from the support and deprotected. Further purification is
usually advised. When the complete operation becomes routine, the synthesis of oligonucleotides
becomes reliable and their biological activity is assured. Since most laboratories are not interested
in this as a research project, the goals are for DNA synthesis to become cheaper, faster, better, easier, and more flexible. This chapter will help you understand the synthesis chemistry and how you
can attain these goals.
The phosphoramidite method of oligonucleotide synthesis is the chemistry of choice for most laboratories because of efficient and rapid coupling and the stability of the starting materials.2 The synthesis is performed with the growing DNA chain attached to a solid support so that excess reagents
which are in the liquid phase can be removed by filtration.3 Therefore, no purification steps are required between cycles. This support material is a form of silica, controlled-pore-glass (CPG)
beads.4 The particle size and the pore size have been optimized for liquid transfer and mechanical
strength. The synthesis cycle is depicted in Figure 6-1. The starting material is the solid support derivatized with the nucleoside which will become the 3'-hydroxyl end of the oligonucleotide. As
shown in Figure 6-2, the nucleoside is bound to the solid support through a linker attached at the
3'-hydroxyl. The 5'-hydroxyl is blocked with a dimethoxytrityl (DMT) group.
The first step of the synthesis cycle is treatment of the derivatized solid support with acid to remove
the DMT group (Figure 6-4). This frees the 5'-hydroxyl for the coupling reaction. (Figure 6-5). An
activated intermediate is created by simultaneously adding the phosphoramidite nucleoside monomer and tetrazole, a weak acid, to the reaction column. The tetrazole protonates the nitrogen of the
phosphoramidite, making it susceptible to nucleophilic attack. This intermediate is so reactive that
addition is complete within 30 seconds. As shown in Figure 6-5, the phosphoramidite is blocked at
the 5'-OH with the dimethoxytrityl group.
The next step, capping, terminates any chains which did not undergo addition. Since the unreacted
chains have a free 5'-OH, they can be terminated or capped by acetylation. These unreacted chains
are also called “failure products”. Capping is done with acetic anhydride and 1-methylimidazole.5
Since the chains which reacted with the phosphoramidite in the previous step are still blocked with
the dimethoxytrityl group, they are not affected by this step. 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 6-7).
6-2
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
The internucleotide linkage is then converted from the phosphite to the more stable phosphotriester.
Iodine is used as the oxidizing agent and water as the oxygen donor. This reaction is complete in
less than 30 seconds (Figure 6-8).
After oxidation, the dimethoxytrityl group is removed with a protic acid, either trichloroacetic or
dichloroacetic acid. The cycle is repeated until chain elongation is complete. 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. The oligonucleotide is cleaved from the support by a one-hour
treatment with concentrated ammonium hydroxide. Ammonia treatment also removes the cyanoethyl phosphate protecting groups. The crude DNA solution in ammonium hydroxide is then treated
at 55°C for 8 to 15 hours to remove the protecting groups on the exocyclic amines of the bases (Figure 6-9).
Note that synthesis can be performed using methyl, or the newer cyanoethyl phosphoramidites.6
These two versions of synthesis monomers differ only by the protecting group on the phosphorous
oxygen. The synthesis chemistry and the resulting oligonucleotide are the same with both. Excellent
results are obtained with either one.7 The primary difference is that when using methyl phosphoramidites, thiophenol treatment is required to deprotect the internucleotide methyl phosphotriester
groups at the end of synthesis. Thiophenol is foul smelling and toxic. In addition, this adds an extra
30-60 minutes to deprotection time. For these reasons, use of cyanoethyl phosphoramidites is
strongly recommended. Methyl phosphoramidites, since they offer no advantage or unique utility,
are no longer available from Applied Biosystems.
Section 6: Chemistry for Automated DNA Synthesis
6-3
Applied Biosystems
Figure 6-1.
6-4
The Synthesis Cycle
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
The Solid Support - CPG
The Model 391 DNA Synthesizer uses a solid phase synthesis chemistry in which the growing DNA
chain remains covalently attached to an insoluble support. All reagents and solvents flow through
the support which is contained within a synthesis column.
The support used for DNA synthesis is Controlled-Pore-Glass (CPG).4 When used with the Model
391 and other Applied Biosystems reagents, CPG produces coupling efficiencies of 98 to 100%, as
measured by the trityl cation assay. This enables the synthesis of oligomers up to 175 bases in
length.8 CPG is a porous, non-swelling particle which is about 150 microns in diameter and has
500Å pores. A wide-pore CPG support (1000Å) is also available and should be used when synthesizing oligonucleotides greater than 60 bases8. CPG is covalently derivatized with one of the four
nucleosides (A, G, C or T), see Figure 6-2. The reactive groups on these nucleosides are blocked or
protected to prevent unwanted side reactions. They are all blocked at the 5'-hydroxyl with a
dimethoxytrityl group. As shown in Figure 6-3, the exocyclic amines on adenosine (A) and cytosine
(C) are protected by a benzoyl group (Abz, Cbz), and the exocyclic amine on guanosine (G) by an
isobutyryl group (Gib). Thymidine does not need a protecting group since the thymine ring does not
participate in the synthesis chemistry.
As shown in Figure 6-2, CPG has a linker attached to its surface via a siloxane bond. All free silanol
groups are capped to prevent side reactions. The support is then derivatized by covalently attaching
the 3'-hydroxyl of the nucleoside to the linker via a succinate ester bond. The bond is base labile
and allows for removal of the DNA from the support with ammonia. After synthesis is complete the
oligonucleotide is quantitatively cleaved with a free 3'-hydroxyl.
The loading of nucleoside as measured by DMT release is 27 to 30 micromoles per gram of 500Å
support. Pre-filled columns contain 0.2 µmol, 1 µmol or 10 µmol of initial nucleoside. The 0.2 µmol
scale provides sufficient amounts of purified oligomers for most applications. The 1 µmol scale is
used when greater quantities of DNA are needed. The 10 µmol scale is useful for physical studies,
such as X-ray crystallography or nmr. Wide-pore CPG supports are only available on the 0.2-micromole level. The loading of nucleoside is lower, about 15 µmol/gm of 1000Å support. For the
synthesis of long oligonucleotides (“bigmers”), it has been shown that a lower loading of nucleoside
and larger CPG pore size are critical requirements for success.
Section 6: Chemistry for Automated DNA Synthesis
6-5
Applied Biosystems
Typical quantities of oligonucleotide obtained from the different synthesis scales are given below:
synthesis cycle
.2µM
crude yield* (O.D.)
(20mer)
20-25
1µM
100-120
Low**
20-25
10µM
800-1000
*Yield figures based on a 20mer sequence. Absorbance measured at 260nm.
Assuming 33 micrograms/O.D. unit. **Low is a low consumption cycle on the
.2gM scale.
With automated synthesis, the DNA is built from 3' to 5'. Before beginning a synthesis, one of four
support-bound nucleosides (A, C, G or T) contained within a column is placed on the instrument.
This nucleoside is the 3'-terminus of the sequence.
Figure 6-2.
6-6
The DMT protected nucleoside is attached to the controlled pore glass (CPG)
support (B = Base, A, G, C, T).
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
Figure 6-3.
Protected exocyclic base amines. Adenosine (A) and cytosine (C) are protected by a
benzoyl group (Bz), and guanosine (G) by an isobutyryl group (Ib). Thyrnidine (T)
does not need a protecting group.
DNA Synthesis Chemistry Cycle
Each cycle of base addition consists of four steps:
1. Detritylation
2. Coupling
3. Capping
4. Oxidation.
These reaction steps are repeated in the above order until all bases are added. Following synthesis,
the DNA chain must be manually cleaved and deprotected from the solid support. Each step will be
discussed in detail.
Section 6: Chemistry for Automated DNA Synthesis
6-7
Applied Biosystems
Detritylation
The first step in oligonucleotide synthesis is to remove the acid-labile, dimethoxytrityl (DMT) protecting group on the 5'-hydroxyl of the support-bound nucleoside. As shown in Figure 6-4 treatment
with the protic acids, trichloroacetic acid (TCA) or dichloroacetic acid (DCA), will deprotect or detritylate the 5'end. This will yield a reactive 5'-hydroxyl which can couple with a phosphoramidite
during the following addition step. The two acids can be used interchangeably and are both available from Applied Biosystems. Dichloroacetic acid is a weaker acid and may yield lower levels of
depurination in the synthesis of long oligonucleotides. However, this observation has not been consistently corroborated. For convenience, in this manual the detritylation acid will exclusively be referred to as TCA.
Detritylation in more detail
Immediately before detritylation, the CPG support is washed with acetonitrile to eliminate traces of
the preceding reagent. Next, TCA (bottle 14) is delivered to the column to cleave the trityl group.
Detritylation under anhydrous conditions, is a reversible reaction. The DMT cation is highly reactive and can re-tritylate any reactive nucleophile. Detritylation is driven to completion by the removal of the DMT cation from the synthesis column. Therefore, detritylation is conducted by
continuous delivery of TCA and elution of the DMT cation. Unlike the other reactions in the cycle,
there is no wait period for detritylation. Any residual TCA must be removed by an acetonitrile wash
to prevent detritylation of the incoming phosphoramidite. If the phosphoramidite monomer becomes detritylated, an unwanted dimer can form in solution and then couple to the support-bound
nucleotide chain. Continued chain propagation would result in some sequences being longer than
the product, making purification difficult.
Following both acetonitrile washes, the remaining solvent is forced out of the column by an argon
reverse flush - argon passes through the column from top to bottom and pushes the liquid to waste.
For a summary of these steps, see Table 6-1.
6-8
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
Table 6-1. Detritylation Steps
STEP DESCRIPTION
PURPOSE
acetonitrile (bottle 18)
delivery to column
wash column and support, remove traces delivery of
preceding reagent
argon reverse flush
remove residual acetonitrile from column
TCA (bottle 14) delivery to detritylate support-bound nucleoside
column
acetonitrile (bottle 18)
delivery to column
wash column, remove traces of TCA
argon reverse flush
remove residual acetonitrile from column
Note
The complete synthesis cycle contains steps to wash and flush the valve blocks and
delivery lines.
Figure 6-4.
Detritylation. TCA or DCA is used to remove the DMT from the 5'end. This leaves a
5' hydroxyl to react with the incoming phosphoramidite in the coupling step.
Quantitating the released trityl cation indicates the step-wise yield and can be used
to monitor the instrument’s performance.
Monitoring the trityl cation
When the dimethoxytrityl, also referred to as DMT or trityl, protecting group is cleaved from the
nucleotide, it exists as a cation (Figure 6-4). When in acid solution, this cation is relatively stable
and produces a brilliant orange color. It has an absorbance maximum at about 498 nm and extinction
Section 6: Chemistry for Automated DNA Synthesis
6-9
Applied Biosystems
coefficient of about 70,000 in most solvents, such as acetonitrile or dichloromethane. It is easily detected and quantitated spectrophotometrically.
To quantitate the trityl cation released at each detritylation step, the column effluent from the TCA
delivery and the subsequent wash with acetonitrile is collected. Instead of the effluent flowing into
the waste bottle, it is channelled through the trityl collection port into tubes in a fraction collector.
The Model 391 cycles provide for the collection of trityl effluent fractions and include a signal to
advance a fraction collector (user provided).
Next, the absorbance of the fractions is measured to quantify the trityl released in each addition cycle. Since the trityl solution is very concentrated, it must be diluted before quantitating or significant
errors in the readings will occur. Typically, the trityl fraction is brought up to a volume of 10 mL
with 0.1 M p-toluenesulfonic acid in acetonitrile. The trityl yield is then used to calculate the coupling efficiency of each addition step by dividing the next step absorbance value from the previous
step value. From this data, an overall stepwise yield can be determined and the expected product
yield can be estimated. The molar amount of DMT cation can be calculated using Beer’s Law:
A = eCl
where: A - absorbance
e - extinction coefficient
C - concentration
l - path length
overall yield = coupling yield number of couplings
For more details, see the trityl assay procedure discussed in User Bulletin 13 (Revised) in the appendix.
Monitoring the trityl cation is very important, but the results must be interpreted with caution. The
trityl assay is only an indirect measure of synthesis efficiency. Certainly, high trityl cation yields
(>98%) must be present for a good synthesis. However, high trityl yields can be present when a poor
synthesis occurs. The reason for this discrepancy is that although this chemistry is highly refined,
it is not perfect. Unwanted side-reactions do occasionally occur. Some of these side-reactions contribute to the trityl cation released each cycle. In particular, a low level of extraneous chain growth
other than the desired oligonucleotide occurs. Sites besides the 5' hydroxyl group participate in coupling (e.g., imperfectly capped sites on the support or branched sites on the nucleic bases).
Other scenarios for imperfect results is if detritylation or capping is incomplete. This could happen
if the synthesizer is not operating correctly, the cycle is not optimized, or the reagents are wet or
impure. These occasions generate species other than the desired product which release the trityl cation. Low trityl cation yields (<98%) always predict a less than optimal synthesis. In practical terms,
6-10
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
trityl yields of less than 95% will not allow identification or purification of even short oligonucleotides. For the above reasons the trityl assay must be viewed only as a preliminary, yet convenient,
monitor of the synthesizer’s performance. The assay is useful to aid in the early detection and diagnosis of instrument related problems. Many laboratories monitor their trityl fractions only by visual
inspection. With experience, a failed synthesis is detected this way. Evaluation of the oligonucleotide by PAGE or HPLC is much more informative than the trityl assay. Synthesizer or reagent
problems can only be adequately diagnosed by these methods, which are direct analyses of the product.
Depurination
Trichloroacetic acid is a very effective protic acid detritylating agent. However, in the presence of
protic acids, amine-protected purines (Abz and Gib) are susceptible to depurination (removal of the
purine from its sugar). The chemical mechanism is initial protonation at N-7 of the purine ring,
causing increased 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. Oligonucleotides which contain apurinic sites are cleaved during the 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). 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. It has been reported that the 5' terminal purine is more
susceptible to depurination than an internucleotide purine with a 3' and a 5' phosphorous.9
The quantity of DNA fragments generated by apurinic ammoniolysis is usually insignificant. If the
synthesis is conducted Trityl On, for purification by OPC or Trityl On HPLC, the 5' end fragment
of apurinic ammoniolysis will bear a DMT group and may complicate purification. In practice however, using the AB reagents and cycles optimized for DNA synthesis, depurination is not detectable
or significant except for long oligonucleotides (>80 bases) which are purine rich near the 3' end.10
To minimize depurination, each treatment with TCA should not be extended beyond the times specified in the cycles.
IMPORTANT Do not stop a synthesis while the DNA is exposed to TCA. Do not increase TCA
delivery times.
Section 6: Chemistry for Automated DNA Synthesis
6-11
Applied Biosystems
Coupling
Phosphoramidites
Phosphoramidites (shown in Figure 6-5) are chemically modified nucleosides which are used as the
building blocks for synthesizing DNA. They are added to the support-bound nucleotide chain one
at a time until all bases in the sequence are coupled. The cyanoethyl phosphoramidite nucleosides
have the following functional groups:
1. A diisopropylamino on a 3' trivalent phosphorous moiety.11 The phosphoramidite is very
stable and is made highly reactive by the activator, tetrazole.
2. A β-cyanoethyl protecting group on the 3' phosphorous moiety. This group prevents side
reactions and aids in solubility of phosphoramidites. It is removed upon completion of the
synthesis by using ammonia. 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 β-elimination reaction to form acrylonitrile and the deprotected
internucleotide phosphodiester group. Acrylonitrile then reacts irreversibly with ammonia to
form 3-aminopropionitrile, an inert compound.
3. A dimethoxytrityl (DMT) protecting group on the 5' hydroxyl. The DMT is removed during
each detritylation step leaving a reactive 5' hydroxyl available for coupling an incoming
phosphoramidite.
4. A benzoyl protecting group on the exocyclic amines of A and C (Abz, Cbz), and an isobutyryl
protecting group on the exocyclic amine of G (Gib). These amide groups prevent side
reactions and are removed upon completion of the synthesis with ammonia. Since thymidine
is unreactive and does not contain an exocyclic amine moiety, it is not protected.
Figure 6-5.
6-12
Structure of guanosine cyanoethyl phosphoramidite
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
Coupling
Before beginning the coupling step, the support is made anhydrous and free of nucleophiles (e.g.
water) by an extensive wash with acetonitrile. Any extraneous nucleophiles will compete with the
support-bound 5' hydroxyls for the activated phosphoramidite and will decrease coupling efficiency. The column is then dried by an argon reverse flush to remove residual acetonitrile. Tetrazole,
the phosphoramidite activator, is next delivered to the column. According to the oligonucleotide sequence, one or more of the phosphoramidites (bottles 1 to 5) and tetrazole (bottle 9) are then simultaneously delivered to the column. Depending on the synthesis cycle, alternate deliveries of
tetrazole and then base-plus-tetrazole are repeated up to 3 times. When these reagents mix, the mild
acid, tetrazole, (pKa = 4.8, Ref.8) transfers a proton to the nitrogen of the diisopropyl group on the
3' phosphorous (See Figure 6-6). This protonated amine makes a very good leaving group upon nucleophilic attack by the tetrazole to form a tetrazolyl phosphoramidite.12 This is the reactive intermediate which forms the internucleotide phosphite with the support bound 5' hydroxyl. A molar
excess of tetrazole ensures that the phosphoramidite will be activated. The excess of phosphoramidite relative to free 5' hydroxyl ensures that the reaction is nearly quantitative (over 98% coupling).
The coupling steps are summarized in Table 6-2.
Mixed sequence probes are synthesized by simultaneous delivery of up to 5 bases (AGCTX) and
tetrazole with near equivalent coupling. The four nucleoside phosphoramidites have slightly different reactivities, as all different molecules must.13
The cyanoethyl phosphoramidites follow the reactivity order of T > G > C > A. 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 cycle, location of the site in the oligonucleotide, age of the
phosphoramidite solutions, etc.They have a range of about 3% because of these variables.
Section 6: Chemistry for Automated DNA Synthesis
6-13
Applied Biosystems
Table 6-2. Coupling Steps
STEP DESCRIPTION
PURPOSE
acetonitrile (bottle 18) delivery
to column
remove nucleophiles, render support anhydrous
argon reverse flush
remove residual acetonitrile, dry column
tetrazole (bottle 9) delivery
to column
deliver activator
tetrazole + phosphoramidite
(bottles 1 to 5) delivery to
column
activate phosphoramidite, begin coupling reaction
tetrazole delivery to column
continue coupling
tetrazole + phosphoramidite
delivery to column
continue coupling
wait
complete coupling reaction
argon reverse flush
remove the tetrazole and phosphoramidite
IMPORTANT These deliveries are critical. Under-delivery causes low coupling efficiency. Overdelivery wastes reagents.
6-14
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
Figure 6-6.
The coupling step. Phosphoramidites and tetrazole are delivered to the column
which contains the support-bound nucleotide. The diisopropylamine is protonated
and displaced by tetrazole. When the 5' OH couples to the phosphorous, a 5' to 3'
internucleotide linkage is created.
Capping
Because coupling is not always quantitative, a small percentage (up to 2%) of support-bound nucleotides can fail to undergo addition. These truncated, or failure sequences, will remain attached
to the support. If they remain in the hydroxyl form, they can propagate in subsequent coupling steps.
Failure sequences with one less base than the product would then be generated making isolation of
the product more difficult. Capping the remaining free hydroxyls by acetylation eliminates this
problem. The capped failure sequences are then prevented from participating in the rest of the synthesis reactions.
To cap, equal volumes and equimolar amounts of two binary reagents, acetic anhydride (bottle 11)
and 1-methylimidazole5, NMI, (bottle 12), are simultaneously delivered to the column. As shown
in Figure 6-7, the reagents mix and create a powerful acetylating agent. The two reagents need to
Section 6: Chemistry for Automated DNA Synthesis
6-15
Applied Biosystems
be segregated since the active acetylating agent is unstable. This agent reacts at the 5' hydroxyls rendering these moieties unreactive for the remainder of the synthesis. The excess reagents are then
removed by an argon reverse flush. The capping time required to acetylate the 1 or 2% 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.
The efficiency of shorter capping time and the capping/oxidation order have been extensively demonstrated in studies at Applied Biosystems.14
Figure 6-7.
Capping of unreacted chains. The capping reagents, acetic anhydride and 1methylimidazole (NMI), are used to terminate unreacted chains by acetylating the 5'
hydroxyl groups.
Oxidation
The newly formed internucleotide linkage is a phosphite (trivalent phosphorous) triester. The phosphite linkage is unstable and is susceptible to acid and base cleavage. Therefore, immediately after
capping, the trivalent phosphite triester is oxidized to a stable pentavalent phosphate triester. This
is shown in Figure 6-8.
Oxidation follows capping to eliminate the possibility of traces of water from the oxidizing solution
causing acetic anhydride to form acetic acid during capping. This would expose the oligonucle-
6-16
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
otides to acid as well as make capping less effective. Also, scientists at Applied Biosystems have
elucidated the complex chemical pathway whereby a small side-reaction, the phosphitylation of the
0-6 position of guanosine can be minimized when capping immediately follows coupling.5,15 The
enzymatic digestion/base composition assay on oligonucleotides made with different cycle order;
capping then oxidation and oxidation then capping, shows markedly different results. The Applied
Biosystems standard; capping then oxidation, gives far less, usually undetectable, amounts of basemodified nucleosides. Cycles with oxidation then capping give high levels of the mutagenic modified nucleoside, 2,6-diaminopurine.
Iodine is used as a mild oxidant in a basic tetrahydrofuran (THF) solution with water as the oxygen
donor. When the iodine-water-pyridine-THF mixture (bottle 15) is delivered to the column, an iodine-pyridine complex forms an adduct with the trivalent phosphorous. This adduct is decomposed
by water with production of a pentavalent phosphotriester internucleotide group. This is an extremely fast reaction, being quantitative in 30 seconds. The iodine solution is removed by an argon
reverse flush and several acetonitrile washes, each followed by an argon reverse flush.
Other oxidizing agents such as sulfur16 can be used in place of oxygen to create nucleotide phosphate analogs or to introduce radioactive atoms.
IMPORTANT Do not stop the synthesis while the phosphorous is unoxidized.
Figure 6-8.
Oxidation of the trivalent phosphorous. The unstable trivalent phosphorous of the
newly formed internucleotide linkage is oxidized to a stable pentavalent phosphorous
using an iodine solution.
Section 6: Chemistry for Automated DNA Synthesis
6-17
Applied Biosystems
Completion of the Synthesis Cycle
Following oxidation, a cycle of nucleotide addition is complete. The 5' terminus of the oligomer is
protected by the dimethoxytrityl group. DNA synthesis continues by removing the 5' trityl and repeating another cycle of base addition. This is done until DNA of specified length has been fully
synthesized.
Immediately after completing the synthesis, a trityl group may still be attached to the 5' terminus,
according to the user’s option. The ending method instructions may be programmed for the trityl
group to remain attached (trityl on) or be cleaved (trityl off). The oligonucleotides are usually detritylated when purifying by gel electrophoresis or ion exchange HPLC. The trityl groups are usually left intact when purifying by OPC or trityl-specific reverse phase HPLC. The ending method
(trityl on or trityl off) is generally chosen before beginning a synthesis.
Manual Deprotection and Cleavage
When the synthesis is finished, the product and capped failure sequences (still attached to the support) exist as phosphate-protected, base-protected phosphotriesters. Complete deprotection is necessary to produce biologically active DNA. In addition, the oligonucleotides must be cleaved from
the support. These steps are performed manually after removing the column from the instrument.
For complete instructions, refer to the Manual Deprotection and Cleavage in Section 2.
Cleavage
Following synthesis, the DNA remains covalently attached to the support. The diester oligonucleotides are then cleaved from the support by a one hour treatment with fresh, concentrated ammonium hydroxide. The double syringe method17 is a convenient technique for cleavage. As seen in
Figure 6-9, the cleavage occurs at the base-labile ester linkage between the linker of the support and
the 3' hydroxyl of the initial nucleoside. The cleaved DNA has a free 3' hydroxyl.
The DNA, now in solution, is collected in a vial fitted with a Teflon-lined cap. The vial contains the
crude mix (product and failure sequences) of base protected oligonucleotides in ammonium hydroxide. Then, the protecting groups on the exocyclic amines of A, G and C must be removed (see Base
Deprotection).
Phosphate Deprotection
The cyanoethyl protecting groups are removed by treatment with ammonium hydroxide. This occurs at the same time as cleavage making phosphate deprotection very quick and simple. See Figure
6-9.
6-18
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
Base Deprotection
The benzoyl and isobutyryl base protecting groups are removed by placing the vial of DNA at 55 ºC
for 8 to 15 hours. This also cleaves the acetyl caps from the failure sequences. Base deprotection is
an ammoniolysis reaction, where ammonia is a nucleophile, attacking the carbonyl of the amide
protecting groups. For effective treatment, use fresh, concentrated ammonium hydroxide during
cleavage. To ensure no decrease in ammonia concentration, store the reagent in a refrigerator, tightly capped. Discard thirty days after opening.
IMPORTANT Use a rightly sealed DNA collection vial that can withstand positive pressure. The vial
must also have a Teflon-lined cap. Rubber-lined caps have contaminants that leach
out of the cap liner during deprotection. Teflon-lined caps can be ordered from
Wheaton; Part Number 240408, size 13-425.
After completing deprotection, cool the ammonium hydroxide-DNA solution on ice to prevent losses from bubbling. Then remove the ammonia by vacuum. Ammonia is much easier to transfer at
lower temperature than at room temperature. When purifying by OPC or trityl-specific reverse
phase HPLC, keep the solution basic to prevent accidental detritylation. To do this, add one drop of
distilled triethylamine every 10 minutes. Also, avoid heating the sample.
If the 5' DMT group has been left on, it can be removed manually by treatment with 80% acetic
acid/water for 20 minutes at room temperature. The acid is then diluted with ethanol and removed
by vacuum, followed by several rinses with ethanol. This procedure is usually done after reverse
phase HPLC or before radioactively labeling the 5' end prior to analysis by gel electrophoresis.
The deprotected, detritylated DNA has a free 5' and 3' hydroxyl and is biologically active. Desalting
and purification may be necessary before use in experiments. An overview of these procedures is
described later in this section. Details can be found in User Bulletin 13 (Revised) in the appendix of
this manual.
Section 6: Chemistry for Automated DNA Synthesis
6-19
Applied Biosystems
Figure 6-9.
Deprotection and cleavage of β-cyanoethyl protected oligonucleotides. Treatment
with concentrated ammonium hydroxide removes the β-cyanoethyl protecting groups
and cleaves the oligonucleotides from the support. The benzoyl and isobutyryl base
protecting groups (X) are removed by heating at 55°C in ammonia for 8 to 15 hours.
Quantitation of the Oligonucleotide
Nucleic acids of any variety are most easily quantitated by UV spectroscopy, measuring at or near
their UV absorbance maxima, about 260 nm. 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 wavelength measurement. A scan of an oligonucleotide will show broad absorbance with a maxima near
260 nm. Using Beer’s law, the concentration of the solution and absolute quantity can be calculated.
As a useful approximation, 1 optical density unit (odu) of single-stranded oligonucleotide consists
of about 33 micrograms, by mass. An approximation to relate absorbance to molar quantities is that
a micromole of oligonucleotide has a number of odu equal to 10 times the number of bases. For
example, a micromole of a 20mer would be 200 odu.
Storage of the Oligonucleotide
Most applications for synthetic oligonucleotides require less DNA than a synthesis provides. Fortunately, oligonucleotides can be stored easily, with little or no degradation for long periods of time.
It is probably most convenient to store them refrigerated as a solution, in either a crude or purified
state. The media may be concentrated ammonia, used to cleave and deprotect the crude oligonucleotide, water, or dilute buffer or salt. Typical aqueous media may contain ethanol, acetonitrile, tri6-20
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
ethylammonium acetate (TEAA), EDTA, etc. It is important to keep the oligonucleotides cold to
minimize degradation and bacterial growth. Alternatively, oligonucleotides may be stored dried as
a pellet in a clean, dry vessel, such as a microcentrifuge tube. The solution used to elute purified
oligonucleotides from OPC, 20% acetonitrile, is a convenient and stable storage media. When
stored by these means, oligonucleotides are stable for over a year. Avoid solutions that are mutagenic, oxidizing or outside the pH range of 3-12.
Analysis and Purification
This section reviews the most common methods for purification of synthetic oligonucleotides.
HPLC (high performance liquid chromatography) and PAGE (polyacrylamide gel electrophoresis)
can provide a high level of purity, but require initial capital investments and are labor intensive and
time consuming. A short oligonucleotide (<30 bases) made with typically high synthesis efficiency
(>98% average DMT yield/cycle) may only require a less stringent purification, with efficient desalting and removal of non-nucleoside synthesis by-products. These methods are elaborated in detail in User Bulletin No. 13 (Revised).
OPC, Oligonucleotide Purification Cartridge
The Oligonucleotide Purification Cartridge (OPC, AB part # 400771) was designed specifically for
rapid, easy purification of synthetic oligonucleotides.18,19 The method is based on a small, syringemounted cartridge containing an adsorbent material with an affinity for DMT oligonucleotides. The
ammonia solution of the crude DMT oligonucleotide (10-20 crude odu) is applied directly to the
cartridge. The DMT oligonucleotide product is retained. By-products, failure sequences not bearing
a DMT group, and other impurities are not retained and are eluted. The DMT group of the OPCbound oligonucleotide is removed with a mild acid solution, then the purified oligonucleotide is
eluted (typically 1-5 odu) with about 1 ml of a 20% acetonitrile solution. The entire operation requires 15-20 minutes, and several OPC purifications can be conducted in parallel. Efficient, reliable
purifications are achieved with oligonucleotides up to about 70 bases.
A recent report has shown that OPC purification is effective with oligonucleotides longer than 70
bases when a preliminary treatment with a lysine solution is conducted, after synthesis and before
ammonia treatment.20 Lysine acts to cleave apurinic sites, a major source of DMT bearing failure
sequences. This purifying operation allows OPC to be effective even with long oligonucleotides.
Several features of this operation are noteworthy. No sample preparation is required. The OPC material is stable to concentrated ammonia. The ammonia solution provides a denaturing media, eliminating secondary structure and hydrogen-bonding, which would allow co-elution of partially
complementary failure sequences. The DMT group is detached and retained in the cartridge. Thus,
Section 6: Chemistry for Automated DNA Synthesis
6-21
Applied Biosystems
the purified, fully deprotected oligonucleotide is eluted in a small volume of 20% acetonitrile in water, completely desalted and ready for use.
PAGE
Polyacrylamide gel electrophoresis (PAGE) is a widely used method for the analysis and purification of oligonucleotides.21 When a mixture of charged molecules are exposed to an electric field,
they will migrate with velocities determined primarily by their mass to charge ratios. This ratio
changes linearly with the log of the molecular weight of DNA of differing chain lengths. This allows a very ordered progression of oligonucleotides with mobility decreasing as the length increases. Proper PAGE technique can provide resolution, and preparative isolation, of single base length
differences.
Oligonucleotide samples for PAGE are typically prepared by drying a known odu amount (by measuring absorbance) and redissolving in media such as formamide/1X TBE: 9/1 or 7M urea, which
are denser than the 1X TBE running buffer. These media and the gel matrix (containing 7M urea)
should be denaturing to ensure disruption of secondary structure and hydrogen-bonding.
The most desirable format for PAGE of oligonucleotides is the slab gel. There are many commercial
devices which essentially consist of a sandwich of two glass plates held apart by two side spacers.
The thickness of the spacers determine the gel thickness. For UV shadowing, .75 - 1.5 mm is convenient. The acrylamide solution is poured in between and allowed to polymerize. A comb is also
inserted, which forms wells where the oligonucleotide is loaded. Each well forms a vertical lane
during the electrophoresis. The plate length should be about 16-40 cm, depending on oligonucleotide length. The width depends only on the number of samples to be run. Dyes, such as bromophenol blue and xylene cyanol are run either in the samples or by themselves as indicators of the
migration distance of the oligonucleotides. The acrylamide concentration of the gel matrix determines the velocity of the oligonucleotides. A range of 8-20% is typical. Longer oligonucleotides
require lower acrylamide concentration.
When the dyes indicate the appropriate migration distance of the oligonucleotide product, the power
is turned off and the gel sandwich is disassembled. There are several methods of analysis: UV shadowing, staining, and radiolabelling/autoradiography.
UV shadowing
This is the simplest, easiest, and introduces the fewest artifacts into the analysis. Depending on oligonucleotide length, from 0.5 to 10 odu are loaded in a well. After electrophoresis, the gel is transferred from the plates to a clear plastic wrapped, fluorescent TLC plate, most conveniently plasticbacked, 20x20cm. The gel is visualized with a UV lamp at short wavelength. The bands appear dark
6-22
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
against a fluorescent green background. A permanent record can be made by photography through
a green filter.
Staining
Many dyes and intercalating agents, such as methylene blue, ethidium bromide, Stains-All, and others, will visualize oligonucleotides in a polyacrylamide matrix. After electrophoresis, the gel is
transferred to a pan containing the staining agent and let soak for some time period. This technique
is more sensitive than UV shadowing, but is more time-consuming and uncertain than UV shadowing.
Radiolabelling
This is the most sensitive, but most laborious method of analysis of oligonucleotides. Typically
about 0.01 odu is phosphorylated enzymatically with T4 polynucleotide kinase and gamma-32P
ATP to give 5'32P phosphorylated oligonucleotides. Alternatively, 35S ATP can be used, and the
oligonucleotides can be labelled at either the 5'or 3' termini. The radiolabelled samples are electrophoresed on the gel. The gel is wrapped in plastic and exposed to X-ray film in a dark room. An
autoradiogram is generated in a time period ranging from minutes to days, depending on the specific
activity of the ATP and other radiolabelling parameters. The film has a finite capacity for development and exposure time must be carefully monitored, usually by taking several exposures. The gel
pattern on the film may be quantitated by densitometry. Product identification is obvious when the
appropriate tracking dye or oligonucleotide standard is also present on the gel.
By any of these techniques, the gel pattern of electrophoresis of an oligonucleotide can be very diagnostic about the course of synthesis. Many synthesizer and reagent problems can be diagnosed
by the appearance and relative amounts of the “failure bands”. Also, PAGE has a distinct advantage
in that many samples can be analyzed and purified concurrently. The equipment is relatively inexpensive and easy to maintain. For bigmers, PAGE is usually the most efficient purification method
and the only analytical method. The primary disadvantages of PAGE are that it is labor intensive
and dependent on good technique.
Oligonucleotides can be purified by locating the product band by UV shadowing and excising the
gel material therein with a clean razor blade. The gel material which is removed should be free of
failure bands, most typically the lower N-1 band. In a preparative electrophoresis run, the product
is run further, on a thicker gel, than in an analytical run, to maximize the separation of the product
band from the N-1 band. The excised gel fragment(s) is soaked in an elution buffer. The gel debris
is then removed by a desalting method, such as with an OPC cartridge.
Section 6: Chemistry for Automated DNA Synthesis
6-23
Applied Biosystems
HPLC
High Performance Liquid Chromatography (HPLC) is another efficient method which combines
quantitative analysis and purification of oligonucleotides. One of the advantages of HPLC is a high
level of automation. Systems are available which allow for repetitive programmed injection, analysis, and data manipulation and storage. Two different types of column adsorbents are popular for
oligonucleotides; reverse-phase and ion-exchange.
Reverse-phase adsorbents discriminate by the hydrophobic differences between oligonucleotides of
varying lengths and sequences. When the 5' DMT is on the oligonucleotide, this group is dominant
in its interaction with the support adsorbent. Reverse-phase columns also can adequately resolve
DMT off oligonucleotides with sufficient capacity. The mobile phase is typically a volatile buffer
such as 0.1M triethylammonium acetate. The oligonucleotides are eluted with a gradient of increasing organic solvent, such as acetonitrile. These conditions are non-denaturing. Occasionally, certain
sequences can exhibit unpredictable HPLC elution patterns, caused by inter- or intramolecular secondary structure and hydrogen-bonding effects.
Ion-exchange adsorbents elute oligonucleotides based on increasing charge, i.e. chain length. An
increasing salt gradient in the mobile phase is used to displace the oligonucleotide phosphate anions. The salt anions, such as ammonium sulfate or sodium phosphate, and the DNA pair with the
adsorbent-bound cation, usually alkylated ammonium species. Since ion-exchange analysis separates only on the basis of increasing charge, the desired product oligonucleotide will always elute
after the lesser charged failure sequences. The high salt mobile phase also provides a denaturing
media. Both of these factors allow easy product identification. Preparative ion-exchange HPLC requires a final desalting operation, most efficiently conducted with OPC.
Consult the chapter on HPLC in User Bulletin 13 (Revised), found in the appendix, for more details.
Alternative Chemistries
In addition to the phosphoramidite chemistry method to prepare “normal” phosphodiester oligonucleotides, recently many alternative chemistries have been demonstrated on AB synthesizers. These
other products include; synthetic RNA, phosphate-analog oligonucleotides, and chemically derivatized oligonucleotides which can be covalently attached to other molecules.
RNA Synthesis
RNA oligoribonucleotides can be synthesized on the Model 391.22,23 Using the 2' silyl 5' DMT cyanoethyl phosphoramidite RNA monomers, the only cycle change is to increase the coupling wait
time to 600 seconds. The RNA monomers bear a large 2' silyl protecting group and therefore require
6-24
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
longer time to couple. The details from Applied Biosystems User Bulletin No. 47 apply for the
Model 391 with a few, simplifying refinements:
The ammonia/ethanol : 3/1 solution of crude RNA oligo (DMT OFF) is heated overnight at 55 ºC,
dried, treated with 10 microliters (per odu of crude RNA oligo) of 1 molar tetrabutylammonium fluoride/THF for 6 hours. An equivalent volume of 0.1M TEAA is added, mixed, and evaporated to
near dryness. 1 ml of 0.1 M TEAA is again added, mixed and applied to an OPC cartridge, following
the desalting protocol:
1. pre-wash the OPC cartridge with 5 ml acetonitrile, then 5 ml TEAA
2. load the RNA oligo, recycling it through the cartridge twice, at 1-2 drops/second
3. wash the OPC cartridge with 5 ml 0.1M TEAA, then 10 ml water
4. elute the desalted oligo (up to 50 ODU) with 1 m1 50% acetonitrile
This OPC procedure replaces a long and troublesome Sephadex column to remove the tetrabutylammonium salts which complicate further analysis and purification. The RNA oligo is then ready
for PAGE or HPLC purification/analysis. RNase degradation is not a significant problem, except
when handling less than 0.1 odu.
Note
RNA synthesis monomers are available from Peninsula Laboratories,
611 Taylor Way, Belmont, CA 94002, USA
Telephone: 1-650-592-5392 FAX: 650-595-4071 PEN LABS BLMT.
Figure 6-10. RNA phosphoramidites
Hydrogen-Phosphonate Chemistry
The hydrogen-phosphonate chemistry is useful and effective for preparing either “normal” phosphodiester oligonucleotides or phosphate-analog oligonucleotides.24 User Bulletin No. 44 provides
full details on using the synthesis reagents and the post-synthesis protocols. Although the cycle is
similar, in that it contains coupling, capping25, and detritylation steps, all reagents26 are different
Section 6: Chemistry for Automated DNA Synthesis
6-25
Applied Biosystems
than those used for the phosphoramidite method. The synthesis efficiency is routinely less than the
phosphoramidite method, typically 95-96% trityl yields. The primary advantage to the hydrogenphosphonate chemistry is the flexibility for one-step, post-synthesis conversion of the internucleoside hydrogen-phosphonate groups to a variety of phosphate species27, such as phosphodiesters,
phosphoramidates, phosphotriesters, and most importantly, the phosphorothioates.
Figure 6-11. hydrogen-phosphonate monomers
Phosphorothioate DNA
When the hydrogen-phosphonate synthesis of an oligonucleotide is done, the column may be removed and a sulfurizing reagent introduced by the double-syringe method.17,28 All the internucleoside hydrogen-phosphonates are rapidly converted to phosphorothioates. After regular ammonia
cleavage/deprotection, oligonucleotides bearing phosphorothioate linkages behave chemically similarly to “normal” phosphodiester DNA. They show the same electrophoresis and HPLC behavior.
This class of phosphate-analog DNA has shown activity in anti-sense translation arrest experiments. In particular, both a sequence, and non-sequence, specific effect has been observed in inhibiting in vitro viral replication of HIV.29
6-26
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
CONVERSION OF HYDROGEN-PHOSPHONATE
TO PHOSPHATE ANALOGS
Figure 6-12.
5' Attachments
In the last several years many applications have been identified and developed for the covalent attachment of small molecules to oligonucleotides. These molecules include fluorescent dyes30,
biotin31, proteins32, and other species that allow the identification of oligonucleotides in biological
systems. In addition, oligonucleotides can be derivatized to allow attachment to solid supports.33 In
this way, for example, an affinity matrix can be constructed to purify a sequence complementary to
the support-bound sequence.
The attachment can be made at several sites on the oligonucleotide. However, this site must not impair hybridization or confer chemical instability. For these reasons and for synthesis ease and efficiency, the 5' terminus is usually the preferred location for derivatization of an oligonucleotide.
Section 6: Chemistry for Automated DNA Synthesis
6-27
Applied Biosystems
Fluorescent-dye Linked Sequencing Primers
Amino Link 2
The 5' fluorescent dye labelled sequencing primers used for the Applied Biosystems 370A DNA
Sequencer consist of three parts; the oligonucleotide primer, a linker, and a fluorescent dye. The
linker bears a highly nucleophilic primary amine group which reacts with the electrophilic N-hydroxy succinimide group of the fluorescent dye. This linker group is created with AMINOLINK 2
(AB Part No. 400808).34 Aminolink 2 is a phosphoramidite molecule with a six carbon chain and
a protected amine group. (Figure 6-13). This reagent is handled and used like a phosphoramidite
nucleoside. Activation with tetrazole forms an active intermediate that couples to the 5' hydroxyl
terminus of the support-bound oligonucleotide, in the final coupling cycle. Oxidation and ammonia
cleavage/deprotection yields the aminolink-oligonucleotide, in solution. Coupling to the fluorescent dye-NHS ester, or other electrophilic species, is conducted in a homogeneous solution.
6-28
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
Figure 6-13. Aminolinked oligonucleotide (in solution)
Section 6: Chemistry for Automated DNA Synthesis
6-29
Applied Biosystems
References
1. Efcavitch, J.W., “Automated System for the Optimized Chemical Synthesis of
Oligodeoxyribonucleotides”, Macromolecular Sequencing and Synthesis, Selected Methods
and Applications, pages 221-234, Alan R. Liss, Inc. (1988).
2. Beaucage, S.L. and Caruthers, M.H., Tetrahedron Letters 22, 1859-1862 (1981).
Beaucage, S.L. and Caruthers, M.H., United States Patent #4,668,777
3. Matteucci, M.D. and Caruthers, M.H., J. Am. Chem. Soc., 103, 3185 (1981)
Matteucci, M.D. and Caruthers, M.H., United States Patent #4,458,066
4. Adams, S.P., Kavka, K.S., Wykes, E.J., Holder, S.B., and Galluppi, G.R., J. Am. Chem. Soc.,
105, 661 (1983).
5. Eadie, J.S. and Davidson, D.S., Nucleic Acids Research, 15, 8333-8349, (1987).
Farrance, I.K., Eadie, J.S., and Ivarie, R., Nucleic Acids Research, 17, 1231-1245, (1989).
6. Sinha, N.D., Biernat, J., and Koster, H., Tetrahedron Letters, 24, 5843-5846 (1983).
7. McBride, L.J., Eadie, J.S., Efcavitch, J.W., and Andrus, W.A., Nucleosides and Nucleotides,
6, 297-300 (1987). Yeung, A.T., Dinehart, W.J. and Jones, B.K., Nucleic Acids Research, 16,
4539-4554 (1988).
8. Efcavitch, J.W., McBride, L.J. and Eadie, J.S., Biophosphates and Their Analogues Synthesis, Structure, Metabolism and Activity, pages 65-70, Bruzik, K.S. and Stec, W.J. (Eds),
Proceedings of the 2nd International Symposium on Phosphorus Chemistry Directed Towards
Biology, Lodz, Poland, 1986.
9. Tanaka, T. and Letsinger, R.L., Nucleic Acids Research, 10, 3249-3260, (1982).
10.Efcavitch, J.W. and Heiner, C., Nucleosides and Nucleotides, 4, 267 (1985).
11. McBride, L.J. and Caruthers, M.H., Tetrahedron Letters, 24, 245-248 (1983).
12.Dahl, O., Phosphorus and Sulfur, 18, 201-204 (1983). Dahl, B.H., Nielsen, J. and Dahl, O.,
Nucleic Acids Research, 15, 1729-1743, (1987).
13.Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Summers, M.F. and Byrd, R.A., Nucleic Acids
Research, 15, 8181-8196, (1985).
14.Applied Biosystems Nucleic Acid Research News, Issue No. 7, October 1988 “The Effect of
the Synthesis Cycle on the Chemical Authenticity of Synthetic DNA”.
15.Bauer, B.F. and Holmes, W.M., Nucleic Acids Research, 15, 812 (1989).
16.Stec, W.J., Zon, G., Egan, W. and Stec, B., J. Am. Chem. Soc., 106, 6077-6079 (1984).
17.The DOUBLE-SYRINGE METHOD for manual ammonia cleavage:
a. Attach an empty luer tip syringe, with plunger fully inserted, into one end of the synthesis
column.
b. Load 2 ml of conc. ammonia in another luer tip syringe and attach to the other end of the
column
6-30
Section 6: Chemistry for Automated DNA Synthesis
Applied Biosystems
c. Holding a syringe in each hand, carefully inject the reagent through the column to the
empty syringe and return the reagent through the column several times.
d. Allow it to stand for at least one hour at room temperature.
e. Drain all of the reagent into one syringe, detach and eject contents carefully into an
appropriate vial for heating to achieve complete deprotection.
18.L.J. McBride, McCollum, C., Davidson, S., Efcavitch, J.W., Andrus, A., and Lombardi, S.J.,
Biotechniques, 6, No. 4, 362-367 (1988) “A New, Reliable Cartridge for the Rapid
Purification of Synthetic DNA”. Applied Biosystems User Bulletin No. 51 “OPC Purification
of Long Oligonucleotides”.
19.“Rapid Purification, Desalting, and Detritylation of Synthetic Oligonucleotides”, McCollum,
C., Davidson, S., McBride, L.J., Efcavitch, J.W., and Andrus, A., poster, FASEB, Las Vegas,
1988.
20.Horn, T. and Urdea, M.S., Nucleic Acids Research, 16, 11559-11571, (1988).
21.Efcavitch, J.W., “The Electrophoresis of Synthetic Oligonucleotides”, Gel Electrophoresis of
Synthetic Oligonucleotides, 2nd Ed., IRL Press, Practical Approach Series, in press.
22.Applied Biosystems User Bulletin No. 47 “RNA Synthesis” April 1988.
23.Usman, N., Ogilvie, K.K., Jiang, M.-Y., and Cedergren, R.J., J. Am. Chem. Soc., 109, 78457854 (1987). Ogilvie, K.K., Damha, M.J., Usman, N., and Pon, R.T., Pure & Applied Chem.,
59, 325-330 (1987). Ogilvie, K.K., Usman, N., Jiang, M, -Y., and Cedergren, R.J., Proc. Natl.
Acad. Sci., 5764 (1988).
24.Froehler, B.C. and Matteucci, M.D. Tetrahedron Letters, 27, 469-472 (1986). Froehler, B.C.,
Ng, P.G., and Matteucci, M.D. Nucleic Acids Research, 14, 5399-5407 (1986). Garegg, P.J.,
Lindh, I., Regberg, T., Stawinski, J., and Stromberg, R., Tetrahedron Letters, 27, 4051-4057
(1986).
25.Andrus, W.A., Efcavitch, J.W., and McBride, L.J., United States Patent #4,816,571,
“Chemical Capping By Phosphitylation During Oligonucleotide Synthesis”.
26.Andrus, A., Efcavitch, J.W., McBride, L.J., and Giusti, B, Tetrahedron Letters, 29, 861-864
(1988).
27.Froehler, B.C., Tetrahedron Letters, 27, 5575-5578 (1986).
28.Andrus, A. and Zon, G., Nucleic Acids Research Symposium Series No. 20, 121-122 (1988).
Applied Biosystems User Bulletin No. 44 “Oligonucleotide Synthesis with HydrogenPhosphonate Monomers”.
29.Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J.S., and Broder, S.,
Proc. Natl. Acad. Sci. USA, 84, 7706-7710 (1987). Stein, C.A., Subsinghe, C., Shinozuka, K.
and Cohen, J.S., Nucleic Acids Research, 16, 3209-3221 (1988).
30.Smith, L.M. et al, Nature, 321, 674-679 (1986).
Section 6: Chemistry for Automated DNA Synthesis
6-31
Applied Biosystems
31.Chu, B.C.F. and Orgel, L.E., DNA, 4, 327-331 (1985). Chollet, A. and Kawashima, E.H.,
Nucleic Acids Research, 15, 1529-1541 (1985). Agrawal, S., Christodoulou, C. and Gait,
M.J., Nucleic Acids Research, 14, 6227-6245 (1986).
32.Li, P., Medon, P.P., Skingle, D.C., Lanser, J.A. and Symons, R.H., Nucleic Acids Research,
15, 5275-5287 (1987). Chu, B.C.F. and Orgel, LE., Nucleic Acids Research, 16, 3671-3691
(1988). Zuckermann, R.N., Corey, D.R. and Schultz, P.G., 1614-1615 (1988). Zuckermann,
R.N. and Schultz, P.G., J. Am. Chem. Soc., 110, 6592-6594 (1988).
33.Ghosh, S.S. and Musso, G.F., Nucleic Acids Research, 15, 5353-5372 (1987).
34.Applied Biosystems User Bulletin No. 49. “Aminolink 2”.
6-32
Section 6: Chemistry for Automated DNA Synthesis
Appendix A: Functions, Cycles and Procedures
MODEL 391 PCR-MATE™ DNA SYNTHESIZER FUNCTIONS
CONTROL
4 - Wait
5 - Advance Fraction Collector
6 - Waste to Port
7 - Waste to Bottle
17 - Interrupt
31 - Recorder On
32 - Recorder Off
33 - Cycle Entry
34 - Cycle End
DELIVER TO COLUMN
9 - #18 to Column
13 - #15 to Column
14 - #14 to Column
19 - Base + Tetrazole to Column
22 - Cap to Column
101 - A to Column
102 - G to Column
103 - C to Column
104 - T to column
105 - X to Column
90 - Tet to Column
106 - #11 to Column
107 - #12 to Column
PRIME DELIVERY LINES
52- A to Waste
53- G to Waste
54 - C to Waste
55 - T to Waste
56 - X to Waste
59 - Cap A to Waste
60 - Cap B to Waste
61 - Tetrazole to Waste
81 - # 15 to Waste
82 - # 14 to Waste
ARGON TO RESERVOIRS
62 - Flush to A
63 - Flush to G
64 - Flush to C
65 - Flush to T
66 - Flush to X
69 - Flush to Tetrazole
70 - Flush to #18
86 - Flush to 14,15
89 - Flush to 11,12
ACETONITRILE
TO RESERVOIRS
RINSE AND FLUSH
1 - Block Flush
2 - Reverse Flush
9 - #18 to Column
10 - #18 to Waste
108 - Flush to Trit
109 - Flush thru Col
PREPARE REAGENTS
16 - Cap Prep
28 - Phosphoramidite Prep
43 - # 18 Prep
51 - Tetrazole Prep
71 - #18 to A
72 - #18 to G
73 - #18 to C
74 - #18 to T
75 - #18 to X
78 - #18 to Tetrazole
84 - #18 to 14
85 - #18 to 15
87 - #18 to 11
88 - #18 to 12
Applied Biosystems
391 FUNCTION LIST
VERSION:
TIME:
DATE:
1.00
04:27
05/11/89
FUNCTION
NUMBER
FUNCTION
NAME
FUNCTION
VALVE LIST
BLOCK FLUSH
REVERSE FLUSH
WAIT
ADVANCE FC
WASTE PORT
WASTE BOTTLE
#18 TO COLM
#18 TO WASTE
#15 TO COLUMN
#14 TO COLUMN
CAP PREP
INTERRUPT
B+TET TO COLUMN
CAP TO COLUMN
PHOS PREP
RCDR ON
RCDR OFF
CYC ENTRY
CYC END
#18 PREP
TET PREP
A TO WASTE
G TO WASTE
C TO WASTE
T TO WASTE
X TO WASTE
CAP A TO WASTE
CAP B TO WASTE
TET TO WASTE
FLUSH TO A
FLUSH TO G
FLUSH TO C
FLUSH TO T
FLUSH TO X
FLUSH TO TET
FLUSH TO #18
#18 TO A
#18 TO G
#18 TO C
#18 TO T
#18 TO X
#18 TO TET
#15 TO WASTE
#14 TO WASTE
#18 TO 14
#18 TO 15
FLUSH TO 14, 15
#18 TO 11
#18 TO 12
FLUSH TO 11, 12
TET TO COLUMN
0, 13, 14, 16
12, 13, 14
1
2
4
5
6
7
9
10
13
14
16
17
19
22
28
31
32
33
34
43
51
52
53
54
55
56
59
60
61
62
63
64
65
66
69
70
71
72
73
74
75
78
81
82
84
85
86
87
88
89
90
A-4
1, 12, 16, 17
1, 13, 17
2, 12, 16, 18
3, 12, 16, 19
22
6, 12, 16, 20, 23
4, 5, 12, 16, 22
20, 23
17
23
11, 13, 20
10, 13, 20
9, 13, 20
8, 13, 20
7, 13, 20
5, 13, 22
4, 13, 22
6, 13, 23
0, 11, 21
0, 10, 21
0, 9, 21
0, 8, 21
0, 7, 21
0, 6
0, 1
1, 11, 17, 21
1, 10, 17, 21
1, 9, 17, 21
1, 8, 17, 21
1, 7, 17, 21
1, 6, 17
2, 13, 18
3, 13, 19
1, 3, 17
1, 2, 17
0, 2, 3
1, 5, 17
1, 4, 17
0, 4, 5
6, 12, 16, 23
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 FUNCTION LIST
VERSION:
TIME:
DATE:
1.00
03:16
05/10/89
FUNCTION
NUMBER
FUNCTION
NAME
92
93
94
95
96
97
98
99
USER A
USER B
USER C
USER D
USER E
USER F
USER G
USER H
FUNCTION
NUMBER
FUNCTION
NAME
FUNCTION
VALVE LIST
101
102
103
104
105
106
107
108
109
A TO COLUMN
G TO COLUMN
C TO COLUMN
T TO COLUMN
X TO COLUMN
#11 TO COLUMN
#12 TO COLUMN
FLUSH TO TRIT
FLUSH THRU COL
11, 12, 16, 20
10, 12, 16, 20
9, 12, 16, 20
8, 12, 16, 20
7, 12, 16, 20
5, 12, 16, 20
4, 12, 16, 22
0, 12, 15
0, 12, 16
Appendix A: Functions, Cycles and Procedures
FUNCTION
VALVE LIST
A-5
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
1
.2µM
63
04:22
05/11/89
FUNCTION
#
NAME
1
10
#18 TO WASTE
2
9
# 18 TO COLM
3
2
REVERSE FLUSH
4
1
BLOCK FLUSH
5
28
PHOS PREP
6
90
TET TO COLUMN
7
19
B+TET TO COLM
8
90
TET TO COLUMN
9
19
B+TET TO COLM
10
90
TET TO COLUMN
11
19
B+TET TO COLM
12
90
TET TO COLUMN
13
4
WAIT
14
16
CAP PREP
15
2
REVERSE FLUSH
16
1
BLOCK FLUSH
17
22
CAP TO COLUMN
18
4
WAIT
19
10
#18 TO WASTE
20
2
REVERSE FLUSH
21
1
BLOCK FLUSH
22
81
#15 TO WASTE
23
13
#15 TO COLUMN
24
10
#18 TO WASTE
25
4
WAIT
26
2
REVERSE FLUSH
27
1
BLOCK FLUSH
28
10
#18 TO WASTE
29
9
#18 TO COLM
30
2
REVERSE FLUSH
31
9
#18 TO COLM
32
2
REVERSE FLUSH
33
9
#18 TO COLM
34
2
REVERSE FLUSH
35
1
BLOCK FLUSH
36
33
CYC ENTRY
37
10
#18 TO WASTE
38
9
#18 TO COLM
39
2
REVERSE FLUSH
40
1
BLOCK FLUSH
41
5
ADVANCE FC
42
6
WASTE PORT
43
82
#14 TO WASTE
(Continued on next page)
A-6
Page
STEP
TIME
A
2
15
5
3
3
3
2
2
2
2
2
2
15
3
5
3
10
5
3
5
3
3
10
5
15
5
3
5
10
5
10
5
10
5
3
1
3
10
5
3
1
1
3
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
2
.2µM
63
04:22
05/11/89
FUNCTION
#
NAME
14
108
14
108
14
108
14
108
14
108
14
108
9
108
7
1
9
2
1
34
Page
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#18 TO COLM
FLUSH TO TRIT
WASTE BOTTLE
BLOCK FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
CYC END
Appendix A: Functions, Cycles and Procedures
STEP
TIME
A
10
1
10
1
10
1
10
1
10
1
10
1
10
8
1
3
20
5
3
1
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
A-7
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
1µM
64
04:23
05/11/89
FUNCTION
#
NAME
1
10
#18 TO WASTE
2
9
# 18 TO COLM
3
2
REVERSE FLUSH
4
1
BLOCK FLUSH
5
28
PHOS PREP
6
90
TET TO COLUMN
7
19
B+TET TO COLM
8
90
TET TO COLUMN
9
19
B+TET TO COLM
10
90
TET TO COLUMN
11
19
B+TET TO COLM
12
90
TET TO COLUMN
13
4
WAIT
14
2
REVERSE FLUSH
15
1
BLOCK FLUSH
16
16
CAP PREP
17
22
CAP TO COLUMN
18
4
WAIT
19
10
#18 TO WASTE
20
2
REVERSE FLUSH
21
1
BLOCK FLUSH
22
81
#15 TO WASTE
23
13
#15 TO COLUMN
24
10
#18 TO WASTE
25
4
WAIT
26
2
REVERSE FLUSH
27
1
BLOCK FLUSH
28
10
#18 TO WASTE
29
9
#18 TO COLM
30
2
REVERSE FLUSH
31
9
#18 TO COLM
32
2
REVERSE FLUSH
33
9
#18 TO COLM
34
2
REVERSE FLUSH
35
1
BLOCK FLUSH
36
33
CYC ENTRY
37
10
#18 TO WASTE
38
9
#18 TO COLM
39
2
REVERSE FLUSH
40
1
BLOCK FLUSH
41
5
ADVANCE FC
42
6
WASTE PORT
43
82
#14 TO WASTE
(Continued on next page)
A-8
Page 1
STEP
TIME
2
15
5
3
3
3
3
2
3
2
3
2
15
5
3
3
12
8
3
5
3
3
10
5
15
5
3
5
10
5
10
5
10
5
3
1
3
10
5
3
1
1
3
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
1µM
64
04:23
05/11/89
FUNCTION
#
NAME
14
108
14
108
14
108
14
108
14
108
14
108
9
108
7
1
2
9
2
1
34
Page 2
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#18 TO COLM
FLUSH TO TRIT
WASTE BOTTLE
BLOCK FLUSH
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
CYC END
Appendix A: Functions, Cycles and Procedures
STEP
TIME
10
1
10
1
10
1
10
1
10
1
10
1
10
8
1
3
1
20
5
3
1
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
A-9
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
Low
61
04:24
05/11/89
FUNCTION
#
NAME
1
10
#18 TO WASTE
2
9
# 18 TO COLM
3
2
REVERSE FLUSH
4
1
BLOCK FLUSH
5
28
PHOS PREP
6
90
TET TO COLUMN
7
19
B+TET TO COLM
8
90
TET TO COLUMN
9
19
B+TET TO COLM
10
90
TET TO COLUMN
11
4
WAIT
12
16
CAP PREP
13
2
REVERSE FLUSH
14
1
BLOCK FLUSH
15
22
CAP TO COLUMN
16
4
WAIT
17
10
#18 TO WASTE
18
2
REVERSE FLUSH
19
1
BLOCK FLUSH
20
81
#15 TO WASTE
21
13
#15 TO COLUMN
22
10
#18 TO WASTE
23
4
WAIT
24
2
REVERSE FLUSH
25
1
BLOCK FLUSH
26
10
#18 TO WASTE
27
9
#18 TO COLM
28
2
REVERSE FLUSH
29
9
#18 TO COLM
30
2
REVERSE FLUSH
31
9
#18 TO COLM
32
2
REVERSE FLUSH
33
1
BLOCK FLUSH
34
33
CYC ENTRY
35
10
#18 TO WASTE
36
9
#18 TO COLM
37
2
REVERSE FLUSH
38
1
BLOCK FLUSH
39
5
ADVANCE FC
40
6
WASTE PORT
41
82
#14 TO WASTE
42
14
#14 TO COLUMN
43
108
FLUSH TO TRIT
(Continued on next page)
A-10
Page 1
STEP
TIME
2
15
5
3
3
3
2
2
2
2
15
3
5
3
10
5
3
5
3
3
10
5
15
5
3
5
10
5
10
5
10
5
3
1
3
10
5
3
1
1
3
10
1
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
Low
61
04:24
05/11/89
FUNCTION
#
NAME
14
108
14
108
14
108
14
108
14
108
9
108
7
1
9
2
1
34
Page 2
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#14 TO COLUMN
FLUSH TO TRIT
#18 TO COLM
FLUSH TO TRIT
WASTE BOTTLE
BLOCK FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
CYC END
Appendix A: Functions, Cycles and Procedures
STEP
TIME
10
1
10
1
10
1
10
1
10
1
10
8
1
3
20
5
3
1
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
A-11
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
A-12
10µM
53
04:25
05/11/89
FUNCTION
#
NAME
1
10
2
9
3
2
4
9
5
2
6
9
7
2
8
1
9
28
10
61
11
19
12
4
13
2
14
1
15
16
16
22
17
4
18
10
19
2
20
1
21
81
22
13
23
4
24
10
25
2
26
1
27
10
28
9
29
2
30
9
31
2
32
9
33
2
34
9
35
2
36
1
37
33
38
10
39
9
40
2
41
1
42
5
43
6
(Continued next page)
Page 1
#18 TO WASTE
# 18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
PHOS PREP
TET TO WASTE
B+TET TO COLM
WAIT
REVERSE FLUSH
BLOCK FLUSH
CAP PREP
CAP TO COLUMN
WAIT
#18 TO WASTE
REVERSE FLUSH
BLOCK FLUSH
#15 TO WASTE
#15 TO COLUMN
WAIT
#18 TO WASTE
REVERSE FLUSH
BLOCK FLUSH
#18 TO WASTE
#18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
CYC ENTRY
#18 TO WASTE
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
ADVANCE FC
WASTE PORT
STEP
TIME
A
3
50
45
50
45
50
45
3
3
6
55
15
45
3
3
40
5
3
45
3
3
50
15
3
45
3
3
50
45
50
45
50
45
50
45
3
1
3
80
45
3
1
1
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
Page 2
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
STEP
NUMBER
44
45
46
47
48
49
50
51
52
53
10µM
53
04:25
05/11/89
FUNCTION
#
NAME
82
14
2
1
10
9
2
1
7
34
#14 TO WASTE
#14 TO COLUMN
REVERSE FLUSH
BLOCK FLUSH
#18 TO WASTE
#18 TO COLM
REVERSE FLUSH
BLOCK FLUSH
WASTE BOTTLE
CYC END
Appendix A: Functions, Cycles and Procedures
STEP
TIME
3
160
45
3
3
80
45
3
1
1
A
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ACTIVE FOR BASES
G
C
T
X
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
A-13
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard #1 BOTTLE CHANGE
9
04:29
05/11/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
1
10
71
62
17
62
52
10
1
A-14
Page 1
BLOCK FLUSH
#18 TO WASTE
#18 TO A
FLUSH TO A
INTERRUPT
FLUSH TO A
A TO WASTE
#18 TO WASTE
BLOCK FLUSH
STEP
TIME
5
7
10
10
1
5
4
7
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Page 1
Standard #2 BOTTLE CHANGE
9
04:29
05/11/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
1
10
72
63
17
63
53
10
1
BLOCK FLUSH
#18 TO WASTE
#18 TO G
FLUSH TO G
INTERRUPT
FLUSH TO G
G TO WASTE
#18 TO WASTE
BLOCK FLUSH
Appendix A: Functions, Cycles and Procedures
STEP
TIME
5
7
10
10
1
5
4
7
10
A-15
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard #3 BOTTLE CHANGE
9
04:29
05/11/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
1
10
73
64
17
64
54
10
1
A-16
Page 1
BLOCK FLUSH
#18 TO WASTE
#18 TO C
FLUSH TO C
INTERRUPT
FLUSH TO C
C TO WASTE
#18 TO WASTE
BLOCK FLUSH
STEP
TIME
5
7
10
10
1
5
4
7
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Page 1
Standard #4 BOTTLE CHANGE
9
04:30
05/11/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
1
10
74
65
17
65
55
10
1
BLOCK FLUSH
#18 TO WASTE
#18 TO T
FLUSH TO T
INTERRUPT
FLUSH TO T
T TO WASTE
#18 TO WASTE
BLOCK FLUSH
Appendix A: Functions, Cycles and Procedures
STEP
TIME
5
7
10
10
1
5
4
7
10
A-17
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard #5 BOTTLE CHANGE
9
04:30
05/11/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
1
10
75
66
17
66
56
10
1
A-18
Page 1
BLOCK FLUSH
#18 TO WASTE
#18 TO X
FLUSH TO X
INTERRUPT
FLUSH TO X
X TO WASTE
#18 TO WASTE
BLOCK FLUSH
STEP
TIME
5
7
10
10
1
5
4
7
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Page 1
Standard #9 BOTTLE CHANGE
6
04:30
05/11/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
1
10
17
61
10
1
BLOCK FLUSH
#18 TO WASTE
INTERRUPT
TET TO WASTE
#18 TO WASTE
BLOCK FLUSH
Appendix A: Functions, Cycles and Procedures
STEP
TIME
5
7
1
4
7
10
A-19
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard #11 BOTTLE CHANGE
7
03:18
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
1
10
17
16
59
10
1
A-20
Page 1
BLOCK FLUSH
#18 TO WASTE
INTERRUPT
CAP PREP
CAP A TO WASTE
#18 TO WASTE
BLOCK FLUSH
STEP
TIME
5
7
1
5
5
7
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Page 1
Standard #12 BOTTLE CHANGE
7
03:18
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
1
10
17
16
60
10
1
BLOCK FLUSH
#18 TO WASTE
INTERRUPT
CAP PREP
CAP B TO WASTE
#18 TO WASTE
BLOCK FLUSH
Appendix A: Functions, Cycles and Procedures
STEP
TIME
5
7
1
5
5
7
10
A-21
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard #14 BOTTLE CHANGE
6
03:19
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
1
10
17
82
10
1
A-22
Page 1
BLOCK FLUSH
#18 TO WASTE
INTERRUPT
#14 TO WASTE
#18 TO WASTE
BLOCK FLUSH
STEP
TIME
5
7
1
5
7
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Page 1
Standard #15 BOTTLE CHANGE
6
03:19
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
1
10
17
81
10
1
BLOCK FLUSH
#18 TO WASTE
INTERRUPT
#15 TO WASTE
#18 TO WASTE
BLOCK FLUSH
Appendix A: Functions, Cycles and Procedures
STEP
TIME
5
7
1
5
7
10
A-23
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard #18 BOTTLE CHANGE
5
03:19
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
1
10
17
10
1
A-24
Page 1
BLOCK FLUSH
#18 TO WASTE
INTERRUPT
#18 TO WASTE
BLOCK FLUSH
STEP
TIME
5
7
1
7
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Page 1
Standard PHOS PURGE
9
03:17
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
28
52
53
54
55
56
61
10
1
PHOS PREP
A TO WASTE
G TO WASTE
C TO WASTE
T TO WASTE
X TO WASTE
TET TO WASTE
#18 TO WASTE
BLOCK FLUSH
Appendix A: Functions, Cycles and Procedures
STEP
TIME
10
4
4
4
4
4
4
7
10
A-25
Applied Biosystems
391 CYCLE/PROCEDURE
NAME:
NUMBER OF STEPS:
TIME:
DATE:
Standard SHUTDOWN
31
03:18
05/10/89
STEP
NUMBER
FUNCTION
#
NAME
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
1
10
71
72
73
74
75
62
63
64
65
66
10
78
87
88
84
85
69
89
86
10
1
17
62
63
64
65
66
70
1
A-26
Page 1
BLOCK FLUSH
# 18 TO WASTE
# 18 TO A
# 18 TO G
# 18 TO C
# 18 TO T
#18 TO X
FLUSH TO A
FLUSH TO G
FLUSH TO C
FLUSH TO T
FLUSH TO X
# 18 TO WASTE
# 18 TO TET
# 18 TO 11
# 18 TO 12
# 18 TO 14
# 18 TO 15
FLUSH TO TET
FLUSH TO 11, 12
FLUSH TO 14, 15
# 18 TO WASTE
BLOCK FLUSH
INTERRUPT
FLUSH TO A
FLUSH TO G
FLUSH TO C
FLUSH TO T
FLUSH TO X
FLUSH TO #18
BLOCK FLUSH
STEP
TIME
5
5
60
60
60
60
60
60
60
60
60
60
10
60
60
60
60
60
60
60
60
10
5
1
5
5
5
5
5
10
10
Appendix A: Functions, Cycles and Procedures
Applied Biosystems
Flow test procedure
STEP
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Appendix A: Functions, Cycles and Procedures
FUNCTION
WAIT
# 18 TO 15
# 18 TO 14
# 18 TO 12
# 18 TO 11
# 18 TO TET
#18 TO X
#18 TO T
#18 TO C
#18 TO G
#18 TO A
#18 TO COLM
# 18 TO WASTE
INTERRUPT
WAIT
PHOS PREP
A TO COL
WAIT
G TO COL
WAIT
C TO COL
WAIT
T TO COL
WAIT
X TO COL
WAIT
TET PREP
TET TO COL
WAIT
CAP PREP
# 11 TO COL
WAIT
# 12 TO COL
WAIT
# 14 TO COL
WAIT
# 15 TO COL
WAIT
# 18 PREP
# 18 TO WASTE
WAIT
#18 TO COL
INTERRUPT
WASTE PORT
TIME
5
40
20
20
20
20
20
20
20
20
20
20
20
1
5
10
120
20
120
20
120
20
120
20
120
20
10
120
20
10
120
20
120
20
120
20
120
20
10
120
20
120
1
1
A-27
Applied Biosystems
Flow test procedure (continued)
STEP
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
A-28
FUNCTION
#14 TO COL
INTERRUPT
BLOCK FLUSH
WASTE BOTTLE
FLUSH TO #18
FLUSH TO 14, 15
FLUSH TO 11, 12
FLUSH TO TET
FLUSH TO X
FLUSH TO T
FLUSH TO C
FLUSH TO G
FLUSH TO A
REVERSE FLUSH
BLOCK FLUSH
TIME
150
1
60
1
60
60
60
60
60
60
60
60
60
60
60
Appendix A: Functions, Cycles and Procedures
Appendix B: DNA Synthesizer Schematic
DNA Synthesizer Plumbing
Diagram
Applied Biosystems
Figure B-1. DNA Synthesizer Schematic
Appendix B: DNA Synthesizer Schematic DNA Synthesizer Plumbing Diagram
B-3
Applied Biosystems
Figure B-2. DNA Synthesizer Plumbing Diagram
B-4
Appendix B: DNA Synthesizer Schematic DNA Synthesizer Plumbing Diagram
Appendix C: Synthesis Log Sheet
Reagent/Solvent Log Sheet
SYNTHESIS LOG SHEET
SEQUENCE NAME:
SEQUENCE:
DATE:
USER:
Nucleoside
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Absorbance of
Trityl Solution
Micromoles
REAGENT/SOLVENT LOG
MONTH:
#1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
NOTES
#2
#3
#4
#5
#9
#11
#12
REAGENT/SOLVENT LOG
MONTH:
#14
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
NOTES
#15
#18
ARGON
WASTE
Appendix D: 391 Illustrated Parts List
Applied Biosystems
Photo
Number
1
2
3
4
5
6
7a
7b
7c
8
9
10
11
12
13
14
14a
14b
15
15a
15b
16
17
Part Number
Description
601379
200023
000951
602279
000592
601440
200019
630008
630003
400790
630004
400501
110026
600513
600574
600309
600511
600792
600497
200325
160003
601861
160010
160005
000198
000276
000928
110003
600507T (old)
Power switch assembly
Rocker switch, on-off
Drip tray
Keyboard display assembly, Model 391
Luer connector, male, 1/4-28, F
Lower luer line, 1/16-in. o.d. x 0.5-mm i.d. x 5-in
Luer connector, 2-in. tube
Bottle, 10 mL
Bottle, 8 oz (200 mL)
Bottle seals, 200 mL, box of 10
Bottle, 16 oz (450 mL)
Bottle seals, 450 mL
Bulkhead union, 1/4 T - 1/4 T (argon inlet)
Particle filter assembly
24-V pressure valve assembly
Mini-vacuum pump assembly
24-V vacuum valve assembly
Vacuum-assist cable assembly
Vacuum ballast assembly
Vacuum switch
Vacuum gage, 30 in. of mercury
Regulator gage assembly
Gage, 0-15 psi
Pressure regulator, 0-15 psi
Vent manifold fitting (3)
Vent manifold sleeve
Waste manifold, 3-port
Elbow, 1/8 MP-1/4T, teflon
Model 381A power supply shown
Voltage Select Modules
17a
17
17
600284
600286
600288
600289
601960 (not shown)
600284
600286
600288
600289
602590 (not shown)
Appendix D: 391 Illustrated Parts List
100-V module
120-V module
220-V module
240-V module
Model 391 power supply
100-V module
120-V module
220-V module
240-V module
Model 391 power supply
D-3
Applied Biosystems
Photo
Number
Part Number
18
19
19a
19b
19c
19d
19e
20
21
22
23
24
25
26
27
254184
254185
254187
254186
254239
600509T
600811
601087
601864
295014
100170
100167
600501
600500
601862
600344
601927
601947
295007
295018
Description
Power Cords
295074
200213
295078
28
29
30
D-4
601854
601929
100176
200215
003020
110019
120 V (50/60 Hz)
220 V (50 Hz)
240 V (50 Hz)
240 V (60 Hz)
100 V (50/60 Hz)
Valve control PCB assembly (analog)
Controller PCB assembly (digital, behind panel)
Cable assembly, 20 pin (to analog PCB)
Cable assembly, 40 pin (to keyboard)
Relays
Battery
+5-V supply
Delivery valve assembly, 8 port (2)
Delivery valve assembly, 4 port
Pressure/vent valve assembly
Solenoid valve assembly, Angar (7)
Base/tetrazole delivery line (6)
Capping/reagent delivery line (5)
Fuse, 0.75 A (all voltage options)
Fuse, 10-A slow-blow, 1/4 in. x 1-1/4 in. (100 V and
120 V): two fuses for part No. 600507T power supply;
two fuses for part No. 601960T power supply
Fuse, 5A, slow-blow (100 V and 120 V): two fuses for
part No. 602590T power supply
Fuse, 5A, slow-blow, 25 mm (220 V and 240 V): two
fuses for part No. 600507T power supply; two fuses for
part No. 601960T power supply
Fuse, 3A, slow-blow (220 V and 240 V); two fuses for
part No. 602590T power supply
PCR-MATE™ memory module, version 1.00
PCR-MATE™ EP memory module, ver.1.00
Fan, 3.15 in. x 3.15 in. x 1.5 in.
Fan filter, 80 mm
Manifold, 8 port, teflon
Plug, 1/4-28 teflon
Appendix D: 391 Illustrated Parts List
Applied Biosystems
Photo
Number
Part Number
Description
Bottle Positions 1-5
31
32
600192
200082
601852
221014
Bottle receptacle assembly (pushbutton type)
Knob, slide, black
Insert/tube assembly, 1-5
O-ring, Kalrez
Bottle Positions 9, 11, 12, and 15
33
34
35
36
110130
601946
001208
003558
003560
002571
37
001208
003559
003560
002571
601945
“Tee,” 1/4-28, teflon (Nos. 11 and 12 pressure)
Insert assembly, 8 oz (2-line)
Ratchet cap receptacle mount (outer shell)
Ratchet receptacle, 8 oz
Ratchet receptacle lid
Wave spring, nickel-plated
Bottle Position 14
38
Ratchet cap receptacle mount (outer shell)
Ratchet receptacle, 16 oz
Ratchet receptacle lid
Wave spring, nickel-plated
Insert assembly, 16 oz (2-line)
Bottle Position 18
39
602458 (not shown)
002268 (not shown)
40
140041 (not shown)
002170 (not shown)
41
601865 (not shown)
Cap assembly, 4 L, 1/8-in. T, 2 line
Gasket, 1.38-in. o.d. x 0.88-in. i.d. x 0.030-in. high,
EPR
Safety carrier, 4 L
Bottle rack, 4 L
Waste Bottle
Appendix D: 391 Illustrated Parts List
Waste cap assembly, 4 L, 1/4-in. T
D-5
Applied Biosystems
Figure D-1. Model 391 PCR-MATE™ DNA Synthesizer, Front View
D-6
Appendix D: 391 Illustrated Parts List
Applied Biosystems
Figure D-2. Model 391 PCR-MATE™ DNA Synthesizer, Left-Side View
Appendix D: 391 Illustrated Parts List
D-7
Applied Biosystems
Figure D-3. Model 391 PCR-MATE™ DNA Synthesizer, Rear View
D-8
Appendix D: 391 Illustrated Parts List
Applied Biosystems
Figure D-4. Model 391 PCR-MATE™ DNA Synthesizer, Controller PCB, Memory Module, and
Cabling
Appendix D: 391 Illustrated Parts List
D-9
Appendix E: Warranty
391 Pre-Installation Manual
Safety First!. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3
User Attention Words, Material Safety Data Sheets, and Waste Profiles . . . . . . . . . . . . . . E-4
Abbreviations, Initializations, and Units. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-6
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7
Chemicals and Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7
Start-Up Chemical Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7
Additional Chemicals and Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-9
Shipping List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-10
User-Supplied Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-11
Site Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-13
Laboratory Space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-13
Electrical Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-14
Power “Quality” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-15
Cooling Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-16
Argon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-16
Ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-16
Liquid Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17
Operator Training at Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17
Proof of Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17
Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17
Preinstallation Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-18
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19
Contacting Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19
To Contact Technical Support by E-Mail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19
Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19
To Contact Technical Support by Telephone or Fax . . . . . . . . . . . . . . . . . . . . . . . . . . E-20
To Reach Technical Support Through the Internet . . . . . . . . . . . . . . . . . . . . . . . . . . . E-22
To Obtain Documents on Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-23
AB LIMITED WARRANTY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-24
Applied Biosystems
Safety First!
Safety is everyone’s concern. Your laboratory has specific safety practices and policies designed to
protect laboratory personnel from the potential hazards, both obvious and hidden, that are present.
The AB service engineer assigned to your laboratory has been trained to safely install your new AB
instrument and to comply with your safety rules. We require our engineers to take the necessary
time to familiarize themselves with the locations of potential hazards, exits, and safety equipment
and steps to be taken during an evacuation or other emergency.
We request that a representative from your laboratory be in the vicinity and available to our engineer at all times while he or she is onsite. We believe, as a minimum, available safety equipment
should consist of a fire extinguisher (Halon™), eye wash, safety shower, eye and hand protection,
adequate ventilation, and protection from sources of radiation (lasers, radioisotopes, contaminated
equipment, radioactive wastes, etc.) that may be present in the area where our engineer will be
working.
We make this request to ensure that both your laboratory and our service personnel have a safe environment to work in during the installation and maintenance of your AB instrument system. Completing this part of the preinstallation procedure as well as the other requirements presented in this
manual will help to ensure both the successful installation and safe use of your equipment.
Appendix E: Warranty 391 Pre-Installation Manual
E-3
Applied Biosystems
User Attention Words, Material Safety Data Sheets, and
Waste Profiles
User Attention Words
Four “user attention words” appear in the text of Applied Biosystems documents. Categorically,
each word implies a particular level of observation or action as follows:
Note
This word is used to call attention to information.
IMPORTANT This information is given because it is necessary for correct operation of the
instrument.
Caution
This word informs the user that damage to the instrument could occur if the
user does not comply with this information.
WARNING
Physical injury to the user or other persons could occur it these required
precautions are not taken.
Material Safety Data Sheets (MSDS)
WARNING
E-4
Some chemicals used with this instrument are hazardous. Hazards are
prominently displayed on the labels of all hazardous chemicals. In addition, the
MSDS, provided in the User’s Safety Information section of this manual, give
information on physical characteristics, hazards, precautions, first aid, spill
clean-up, and disposal procedures. Please familiarize yourself with
information in the MSDSs before handling the reagents or operating the
instrument. Additional free copies of MSDSs are available from Applied
Biosystems upon request.
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Waste Profiles
WARNING
Some chemicals collected in the waste bottle after a normal cycle may be
hazardous and/or require special handling. The waste profiles, presented in
the User’s Safety Information section of this manual, give percentage
compositions of the reagents and inform the user about waste disposal, which
must always be in accord with local, state, and federal regulations. Additional
free copies of the waste profiles are available from Applied Biosystems on
request.
Appendix E: Warranty 391 Pre-Installation Manual
E-5
Applied Biosystems
Abbreviations, Initializations, and Units
AB
Btu/h
CGA
cm
DNA
ea
EP
fpm
ft
g
gal
h
HPLC
Hz
i.d.
in.
kg
kPa
kVA
L
lb
M
m
m3
mL
mm
mol. wt
MSDS
nm
No.
o.d.
oz
P/N
par.
PCR
ppm
psi
V
ºC
ºF
µm
µmol
E-6
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Applied Biosystems
British thermal units/hour
Compressed Gas Association
centimeter
deoxyribonucleic acid
each
extended programmability
feet per minute
feet
gram
gallon
hour
high performance liquid chromatography
hertz
inside diameter
inch
kilogram
kiloPascal
kilovolt-amperes
liter
pound
molar
meter
cubic meters
milliliter
millimeter
molecular weight
material safety data sheet
nanometer
number
outside diameter
ounce
part number
paragraph
polymerase chain reaction
parts per million
pounds per square inch
volt
degrees Celsius
degree Fahrenheit
micron
micromole
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Introduction
This manual will help you prepare for the installation of your Model 391 or Model 391 with extended programmability (EP) DNA Synthesizer. The first part describes (1) items that will be shipped
with the instrument and (2) chemicals and equipment you should obtain before you receive the instrument. The second part of the manual describes site preparation. Careful attention to these requirements will simplify the installation procedure and ensure that the instrument works correctly.
The site preparation section concludes with a Preinstallation Checklist.
The Model 391/391 EP is available with four voltage options. It is important that the internal
voltage setting for your instrument agree with your site voltage. Please see “Shipping List” on
page 10 for specific information.
If you purchased an Applied Biosystems installed instrument:
Please do not unpack the crate. until your local Applied Biosystems service representative arrives
to make the installation. The Start-Up Chemical Kit will be shipped separately and should be unpacked upon arrival. This kit contains sufficient chemicals and reagents to demonstrate that the instrument is operating correctly. If additional DNA will be synthesized immediately following
installation, additional chemicals should be ordered.
If you purchased a user installed instrument (U.S. only):
The User Installation Manual will be sent to you, Please be aware that you will need to order reagents for the installation. We recommend that you order the Start-Up Chemical Kit (P/N 400971),
which contains sufficient chemicals and reagents for demonstrating that the instrument operates
correctly. The kit contains additional items for post-synthesis processing of the oligonucleotide. If
additional DNA will be synthesized immediately following installation, additional chemicals
should also be ordered.
Chemicals and Accessories
Start-Up Chemical Kit
If an Applied Biosystems service engineer will install your Model 391 or 391 EP, a Start-Up Chemical Kit will be sent to you for the installation. The kit contains chemicals and reagents (enough for
at least 100 base additions), four synthesis columns, and a manual deprotection kit. The Start-Up
Chemical Kit will be used during installation to verify instrument performance.
Appendix E: Warranty 391 Pre-Installation Manual
E-7
Applied Biosystems
If you have purchased a user-installed instrument (U.S. only), you must order the Start-Up Chemical Kit, P/N 400971.
The contents of the Start-Up Chemical Kit are listed below:
Part No.
Description
Quantity
001753
Seal, 16-oz bottle
2
002267
Seal, 8-oz bottle
5
200140
Septum, 13 mm, rubber
2
400257
Manual deprotection kit
1
400330
CE-phosphoramidite A, 0.5 g
1
400331
CE-phosphoramidite G, 0.5 g
1
400332
CE-phosphoramidite C, 0.5 g
1
400333
CE-phosphoramidite T, 0.5 g
1
400443
Acetonitrile, 4 L, Burdick & Jackson
1
400445
Trifluoroacetic acid, 10 mL
1
400613
Triethylamine acetate, 2 M, 200 mL
1
400771
Oligo purification cartridge
1
400852
400953
Kit, B-CE large bottle reagents (includes the following items)
1
400060
Anhydrous acetonitrile, 30 mL
2
400236
Trichloroacetic acid, 450 mL
2
400501
16-oz bottle seals, 10/pkg
1
400606
Tetrazole/acetonitrile, 180 mL
1
400607
Acetic anhydride, 180 mL
1
400753
Iodine/water/pyridine, 200 mL
1
400785
1-methylimidazole, 180 mL
1
400790
200-mL bottle seals, 10/pkg
1
Small-scale A CPG column
1
400954
Small-scale C CPG column
1
499955
Small-scale G CPG column
1
400956
Small-scale T CPG column
1
Note
E-8
The only additional reagent you will need for installation is concentrated analytical
grade ammonium hydroxide. Because ammonium hydroxide is a common laboratory
reagent, Applied Biosystems does not supply it in some countries, including the
United States. If you do not have ammonium hydroxide in your laboratory, you can
order it from Baker Laboratories (Concentrated reagent, 29.8%, Baker P/N 9721.1).
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
The Start-Up Chemical Kit includes a manual deprotection kit (P/N 400257) that contains the following:
Part No.
Description
200243
1-mL disposable syringes with luer fitting
Quantity
10
200244
1.5-in. disposable needles with female luer
10
200245
No. 10 rubber stopper
1
110127
Male-to-male luer connector (Alltech 86506CTFE)
5
400048
DNA collection vial
10
140048
Teflon lined vial cap
10
With the exception of the No. 10 rubber stoppers and the luer connectors, this equipment is used on
a one-time basis and should be ordered in sufficient quantities for regular use. In addition, when
possible, it is convenient to carry out the deprotection on several compounds simultaneously, necessitating additional male-to-male luer connectors.
Additional Chemicals and Columns
The supplies included in the Start-Up Chemical Kit will not last beyond the installation period. It is
strongly recommended that an additional supply be ordered before installation. If you wish to order
chemicals and columns for additional syntheses, please contact your local salesperson or call Applied Biosystems. All phosphoramidites, reagents, and synthesis columns should be ordered from
Applied Biosystems with two exceptions:
• Ammonium hydroxide for the cleavage/deprotection step. Order concentrated analytical
grade reagent. Purchase it in 500-mL bottles, keep it sealed tightly, and store it at 4 °C. Do not
use it for more than two weeks after you have opened a bottle.
• Acetonitrile (HPLC-grade) for bottle position 18. The Model 391 or 391 EP is equipped to
handle a 4-L bottle. The water content must be less than or equal to 300 ppm (0.003%). We
have had success with Burdick & Jackson acetonitrile (B&J P/N 015-4).
If you are unsure of the water content of a local supplier’s acetonitrile, or if you know that the water
content is too high, you can control or avoid this problem by (1) checking the acetonitrile by Karl
Fischer titration or (2) distilling HPLC-grade acetonitrile over P2O5. Redistill over CaH2 to remove
acid.
Appendix E: Warranty 391 Pre-Installation Manual
E-9
Applied Biosystems
Shipping List
The instrument will be shipped in a crate with the following accessory items:
Part No.
Description
Quantity
000160
Ferrule, 1/16 in., teflon
2
000161
Bushing, 1/16 in., plastic
2
002170
4-L Bottle
1
002268
Gasket, EPR
1
100072
Wrench, 1/4 in. x 5/16 in.
1
110005
Connector, 1/4 MP-1/4 T, brass
1
110096
Nut, 1/4 in. brass
1
110097
Ferrule, swage, 1/4 in.
1
140024
4-L waste bottle
1
140041
4-L bottle safety carrier
1
200270
Solvent inlet filter, 40 µm
1
221014
O-ring, Karlez
1
225016
Tubing, 1/4-in. o.d., polypropylene
225075
Tubing, Tygon
10 ft (3 m)
400501
Bottle seals, 16 oz, 10/pkg
1
400790
Bottle seals, 200 ml, 10/pkg
1
600223
Trityl collection tubing assembly
1
600235
Fraction collector cable
1
601865
4-L waste cap assembly
1
3 in. (76 mm)
In addition a plug/voltage kit from the list below will be included with your instrument.
Part No.
Description
Voltage and Frequency
401202
Plug/voltage kit for US/Canada
120V (60 Hz)
401203
Plug/voltage kit for Continental Europe
220V (50 Hz)
401204
Plug/voltage kit for United Kingdom
240V (50 Hz)
401205
Plug/voltage kit for Australia
240V (50Hz)
401201
PIug/voltage kit for Japan
100V (50/60Hz)
Verify that the plug/voltage kit you received is the correct one for your installation site.
E-10
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
The 391 EP PCR-MATE will be shipped with these additional items.
Part No.
Description
254125
RS232 cable, 6 ft (1.8 m)
Quantity
1
254132
Null modem
1
254136
RS232 cable, 10 ft (3 m)
1
602022
PC syncom disk
1
User-Supplied Equipment
All customer-supplied items (for trityl analysis and post-synthesis processing of your oligonucleotides) are listed below and should be obtained before the installation. The two most critical items
are a fraction collector and a spectrophotometer. Although measuring trityl cation release is only
an approximate indicator of synthesis success, it is a convenient assay, and so we use it rather than
more meaningful HPLC or capillary electrophoresis analysis to confirm instrument performance.
The following items are supplied by the user:
• Fraction Collector
To monitor synthesis efficiency, a fraction collector must be adjacent to the synthesizer to
collect the trityl solution from the trityl collection port. Any commercially available fraction
collector with an external advance switch will suffice. We recommend that at least one
hundred 10-15 mL volumetric tubes be available. The fraction collector cable and trityl
delivery line are packed with the instrument. The fraction collector must be placed within 30
cm (12 in.) of the left side of the instrument.
WARNING
The trityl effluent is composed of trichloroacetic acid and acetonitrile. Before
you handle chemicals for the instrument, read the material safety data sheets
(MSDS) and waste profile in Appendices A and B of this preinstallation manual.
Always follow the safety precautions (eye protection, clothing, etc.) presented
in the MSDS. Dispose of waste in accord with all local, state, and federal health
and environmental regulations and laws.
• Spectrophotometer
To measure absorbance of the diluted trityl solutions you will need a visible
spectrophotometer capable of reading at a wavelength of 530 nm. Also required are 1-cm
cuvettes and a 0.1-M toluene sulfonic acid/acetonitrile solution for dilution.
• Accessories for Reconstituting Phosphoramidites
1. Glass syringes (2, 5 or 10 mL) kept in an oven at 100-110 °C (212-230 °F). These are used to
dispense anhydrous acetonitrile into the phosphoramidite bottles.
Appendix E: Warranty 391 Pre-Installation Manual
E-11
Applied Biosystems
2. Syringe needles (20 gage) that fit the glass syringes and are 5-10 cm (2-4 in.) long
(Aldrich P/N Z11-711-0).
3. Rubber septa are used to seal the 30-mL bottles of anhydrous acetonitrile
(Aldrich P/N Z10-074-9).
• Heating Bath or Block
The laboratory must have access to a heating bath or block to complete the manual base
deprotection of the synthetic oligonucleotides. The temperature of this bath or block is
typically set at 55-60 °C (131-140 °F).
• Sample Concentrator
The ammonium hydroxide deprotection solution is evaporated before they DNA is used or
purified. Any device capable of removing an aqueous solution is acceptable.
• Toluenesulfonic Acid
Purchase the 500-g bottle of p-toluenesulfonic acid monohydrate, 99% (P/N T-3751) from
Sigma Chemical Co., St. Louis, MO 63178. This is used to dilute the trityl cation.
WARNING
P-toluenesulphonic acid is hazardous and must be handled with great caution.
Refer to the Sigma Chemical Co. MSDS to familiarize yourself with its hazards
and the precautions that must be taken. All procedures must be conducted in
a correctly operating fume hood. Use of eye protection, appropriate rubber
gloves, and safety clothing is imperative.
• Ammonium Hydroxide
As mentioned under “Start-Up Chemical Kit” on page 7, you must purchase concentrated
analytical grade ammonium hydroxide (preferably in 500-mL bottles) for deprotection. This
reagent is not included in the Start-Up Chemical Kit.
WARNING
E-12
Ammonium hydroxide is hazardous and must be handled with great caution.
Refer to the supplier’s MSDS to familiarize yourself with its hazards and the
precautions that must be taken. All procedures must be conducted in a
correctly operating fume hood. Use of eye protection, appropriate rubber
gloves, and safety clothing is imperative.
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Site Preparation
Laboratory Space
Allocating sufficient laboratory space (Figure E-1) is critical for successful installation and troublefree maintenance. A sturdy freestanding table with locking wheels is strongly preferred over a lab
bench because it allows access to the rear of the instrument. Specifically, we recommend an open
bench with plastic laminate top, which can be ordered from Bay Products, 8701 Torresdale Avenue,
Philadelphia, PA 19136 (800) 523-3434 or (215) 338-7300. The Bay Products catalog number is
6303B34.
Sufficient room must be provided for the user-supplied external fraction collector and the 4-L acetonitrile bottle container which will be positioned to the left of the synthesizer. A space within approximately 5 ft (1.5 m) of the instrument must also be provided for safely securing an argon gas
cylinder (size 1A). The dimensions and height of the Model 391 or 391 EP are as follows:
Width
Depth
Height
Weight
52 cm
38 cm
41 cm
29 kg
(20.5 in.)
(15.0 in.)
(16.0 in.)
(65 lb)
Figure E-1. Laboratory Space Requirement
Appendix E: Warranty 391 Pre-Installation Manual
E-13
Applied Biosystems
Electrical Requirements
Power
The Model 391 or 391 EP requires a dedicated electrical line with a circuit breaker (Figure E-2) and
a power rating of at least 1.25 kVA. The outlet must be located close to the instrument. The supplied
power cord is 8 ft (2.5 m).
Grounding
Certain types of electrical noise are exaggerated by poor or improper electrical round connections.
To prevent these problems, the installation site should have a dedicated 1.25-kVA power line and
isolated ground between the instrument and building main electrical service, as depicted in Figure
E-2.
E-14
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Figure E-2. Dedicated Power Line
Power “Quality”
Line Voltage Fluctuation
The line voltage must be within 5% of the nominal figures. High or low voltage conditions will have
an adverse effect on electronic components of the instrument. In areas where the supplied power is
subject to fluctuations exceeding the above limits, a power line regulator is strongly suggested.
Please consult your local Applied Biosystems service representative if you suspect a problem.
Voltage Spikes
Short-duration, high-voltage spikes can be caused by other devices (such as large motors) using the
same power source or by outside influences such as lightning. If your laboratory environment contains devices that are electronically “noisy,” or if you are in an area with frequent electrical storms,
a line conditioner may greatly enhance reliability of your system. The line conditioner must have a
rating of at least 0.5 kVA. Several sources of line conditioners are as follows:
• Best Power, P. 0. Box 280, Necedah, WI 54646 - Tel: (608) 565-7200
• RTE Deltec, 2727 Kurtz St, San Diego, CA 92120 - Tel: (619) 291-4211
• Elgar, 9250 Brown Deer Rd, San Diego, CA 92121 - Tel: (619) 450-0085
Appendix E: Warranty 391 Pre-Installation Manual
E-15
Applied Biosystems
Cooling Requirements
The Model 391 or 391 EP generates a maximum of 600 Btu/h. While there are no special cooling
requirements, a constant laboratory temperature between 16-22 °C (60-72 °F) is recommended.
Avoid placing the instrument close to heaters or cooling ducts. Temperatures below 16 ºC (60 ºF)
must be avoided because they will cause the tetrazole to precipitate from solution.
Argon
A regulated cylinder (size 1A) of prepurified (99.998%) argon must be connected to the rear panel
of the synthesizer. The instrument has a gas input that accepts 1/4-in.-o.d. Parflex™ tubing with appropriate Swagelok® tube fittings (1/4-in. nut and ferrule).
A 10-ft length of Parflex™ tubing (1/4-in. o.d.) and the necessary Swagelok® fittings to connect this
tubing to the instrument are provided.
The argon tank requires a two-gauge regulator with a CGA (Compressed Gas Association) 580 argon cylinder adapter on the inlet side and a Swagelok® fitting that accepts a 1/4-in.-o.d. tube. The
primary gauge (0-3000 psi; 0-25,000 kPa recommended) measures tank pressure, and the secondary
gauge (0-200 psi; 0-2000 kPa recommended) measures regulated pressure. The regulator must be
obtained by the user prior to installation.
Typical argon consumption is approximately 3.0 m3 every four weeks. Size 1A cylinders have a
capacity of 8.2 cm3 and should last approximately three months. The tank should be regulated to
approximately 65 psi (450 kPa).
WARNING
Compressed gas cylinders must be safely attached to the wall or bench by
means of approved restraints. Failure to do so could cause the cylinders to fall
over and explode, which could result in physical harm. Turn off or cap the
cylinders when they are not in use.
Ventilation
Gaseous waste is vented from the waste bottle. Your facility must be equipped with a ventilated laboratory chemical fume hood or the equivaent to remove harmful vapors. The waste vent should be
routed to the chemical exhaust system by one of the methods shown in the User’s Safety Information section at the beginning of this manual.
E-16
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Liquid Waste Disposal
WARNING
Please review the waste profile for information on contents of the Model 391
and 391 EP liquid waste so that it can be disposed of in accordance with all
local, state, and federal health and environmental regulations and laws. Please
also read the MSDS to determine the protective equipment required to work
with and dispose of these chemicals. The waste profile and MSDS are in the
User’s Safety Information section at the beginning of this manual.
Operator Training at Installation
(Applied Biosystems Service Installed Instruments Only)
An important part of in-laboratory installation is operator training. Those persons who will be operating the Model 391 or 391 EP should set aside two uninterrupted days to work with the Applied
Biosystems service representative. If this is not possible, the installation should be rescheduled.
Proof of Performance
Unless previous arrangements have been made, an oligonucleotide of less than 25 bases will be synthesized to demonstrate instrument performance. It is necessary to have the fraction collector, spectrophotometer, and p-toluenesulfonic acid solution prepared so that the trityl assay can be
performed. The passing on-site specification is 98.0% (±0.5%) average coupling yield.
Printer
A printer option consisting of a Hewlett-Packard Think Jet™ printer and interface cable is available
from Applied Biosystems. The appropriate part number depends on the site voltage as follows: P/
N 400815, 100 Vac; P/N 400816, 120 Vac; P/N 400817, 220 Vac; and P/N 400818, 240 Vac. The
interface cable (P/N 100157) may be ordered separately. Although the printer uses any standard 81/2 x 11-in. (European size A4) single or fanfold paper, best print quality is ensured by using H-P
specified Ink Jet paper. Additional paper stock is available from Hewlett-Packard:
Part No. 92261 M: Ink Jet paper, 500 sheets, single sheets
Part No. 92261 N: Ink Jet paper, 2500 sheets, fanfold
Appendix E: Warranty 391 Pre-Installation Manual
E-17
Applied Biosystems
Preinstallation Checklist
The following checklist is designed to ensure that all necessary preparations have been made prior
to installation of the Model 391 or 391 EP. If there are any questions concerning the installation
procedure or preparations, please contact your local Applied Biosystems service representative.
Check (✔)
if ready
1.
Preinstallation manual read and understood
Chemicals and Equipment:
2.
Correct plug/voltage kit_ received
3.
Start-Up Chemical kit received
4.
Additional reagents, including concentrated ammonium hydroxide and
acetonitrile (4 L) ordered.
5.
Fraction collector
6.
Spectrophotometer
7.
Accessories for reconstituting phoshoramidites
8.
Heating block
9.
Sample concentrator
10.
Toluenesulfonic acid
11.
All material safety data sheets (MSDS) and waste profile material read and
understood
Site Preparation:
E-18
12.
Correct laboratory space
13.
Constant laboratory temperature
14.
Argon cylinder, size 1A (regulator and adapter)
15.
Correct ventilation
16.
Waste disposal method established
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Technical Support
Contacting Technical Support
You can contact Applied Biosystems for technical support by telephone or fax, by e-mail, or
through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of
analysis, and other related documents 24 hours a day. In addition, you can download documents in
PDF format from the Applied Biosystems Web site (please see the section “To Obtain Documents
on Demand” following the telephone information below).
To Contact Technical Support by E-Mail
Contact technical support by e-mail for help in the following product areas:
Product Area
E-mail address
Genetic Analysis (DNA Sequencing)
[email protected]
Sequence Detection Systems and PCR
[email protected]
Protein Sequencing,
Peptide and DNA Synthesis
[email protected]
Biochromatography, PerSeptive DNA, PNA and
Peptide Synthesis systems, CytoFluor®, FMAT™,
Voyager™, and Mariner™ Mass Spectrometers
[email protected]
LC/MS
(Applied Biosystems/MDS Sciex)
[email protected]
or
[email protected]
Chemiluminescence (Tropix)
[email protected]
Hours for Telephone Technical Support
In the United States and Canada, technical support is available at the following times:
Product
Hours
Chemiluminescence
8:30 a.m. to 5:30 p.m. Eastern Time
Framingham support
8:00 a.m. to 6:00 p.m. Eastern Time
All Other Products
5:30 a.m. to 5:00 p.m. Pacific Time
Appendix E: Warranty 391 Pre-Installation Manual
E-19
Applied Biosystems
To Contact Technical Support by Telephone or Fax
In North America
To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below.
(To open a service call for other support needs, or in case of an emergency, dial 1-800-831-6844
and press 1.)
Product or
Product Area
Telephone
Dial...
Fax
Dial...
ABI PRISM® 3700 DNA Analyzer
1-800-831-6844,
then press 8
1-650-638-5981
DNA Synthesis
1-800-831-6844,
then press 21
1-650-638-5981
Fluorescent DNA Sequencing
1-800-831-6844,
then press 22
1-650-638-5981
Fluorescent Fragment Analysis (includes GeneScan®
applications)
1-800-831-6844,
then press 23
1-650-638-5981
Integrated Thermal Cyclers (ABI PRISM ® 877 and
Catalyst 800 instruments)
1-800-831-6844,
then press 24
1-650-638-5981
ABI PRISM ® 3100 Genetic Analyzer
1-800-831-6844,
then press 26
1-650-638-5981
BioInformatics (includes BioLIMS, BioMerge™, and
SQL GT™ applications)
1-800-831-6844,
then press 25
1-505-982-7690
Peptide Synthesis (433 and 43X Systems)
1-800-831-6844,
then press 31
1-650-638-5981
Protein Sequencing (Procise Protein Sequencing
Systems)
1-800-831-6844,
then press 32
1-650-638-5981
PCR and Sequence Detection
1-800-762-4001,
then press 1 for PCR,
2 for the 7700 or 5700,
6 for the 6700
or dial 1-800-831-6844,
then press 5
1-240-453-4613
Voyager MALDI-TOF Biospectrometry and Mariner
ESI-TOF Mass Spectrometry Workstations
1-800-899-5858,
then press 13
1-508-383-7855
Biochromatography (BioCAD Workstations and
Poros Perfusion Chromatography Products)
1-800-899-5858,
then press 14
1-508-383-7855
Expedite Nucleic acid Synthesis Systems
1-800-899-5858,
then press 15
1-508-383-7855
Peptide Synthesis (Pioneer and 9050 Plus Peptide
Synthesizers)
1-800-899-5858,
then press 15
1-508-383-7855
PNA Custom and Synthesis
1-800-899-5858,
then press 15
1-508-383-7855
E-20
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
Product or
Product Area
Telephone
Dial...
Fax
Dial...
FMAT 8100 HTS System and Cytofluor 4000
Fluorescence Plate Reader
1-800-899-5858,
then press 16
1-508-383-7855
Chemiluminescence (Tropix)
1-800-542-2369 (U.S.
1-781-275-8581
only),
or 1-781-271-0045
Applied Biosystems/MDS Sciex
1-800-952-4716
1-650-638-6223
Telephone
Dial...
Fax
Dial...
Outside North America
Region
Africa and the Middle East
Africa (English Speaking) and West Asia (Fairlands,
South Africa)
27 11 478 0411
27 11 478 0349
South Africa (Johannesburg)
27 11 478 0411
27 11 478 0349
Middle Eastern Countries and North Africa (Monza,
Italia)
39 (0)39 8389 481
39 (0)39 8389 493
Eastern Asia, China, Oceania
Australia (Scoresby, Victoria)
61 3 9730 8600
61 3 9730 8799
China (Beijing)
86 10 64106608
86 10 64106617
Hong Kong
852 2756 6928
852 2756 6968
Korea (Seoul)
82 2 593 6470/6471
82 2 593 6472
Malaysia (Petaling Jaya)
60 3 758 8268
60 3 754 9043
Singapore
65 896 2168
65 896 2147
Taiwan (Taipei Hsien)
886 2 22358 2838
886 2 2358 2839
Thailand (Bangkok)
66 2 719 6405
66 2 319 9788
Europe
Austria (Wien)
43 (0)1 867 35 75 0
43 (0)1 867 35 75 11
Belgium
32 (0)2 712 5555
32 (0)2 712 5516
Czech Republic and Slovakia (Praha)
420 2 61 222 164
420 2 61 222 168
Denmark (Naerum)
45 45 58 60 00
45 45 58 60 01
Finland (Espoo)
358 (0)9 251 24 250
358 (0)9 251 24 243
France (Paris)
33 (0)1 69 59 85 85
33 (0)1 69 59 85 00
Germany (Weiterstadt)
49 (0) 6150 101 0
49 (0) 6150 101 101
Hungary (Budapest)
36 (0)1 270 8398
36 (0)1 270 8288
Italy (Milano)
39 (0)39 83891
39 (0)39 838 9492
Norway (Oslo)
47 23 12 06 05
47 23 12 05 75
Poland, Lithuania, Latvia, and Estonia (Warszawa)
48 (22) 866 40 10
48 (22) 866 40 20
Portugal (Lisboa)
351 (0)22 605 33 14
351 (0)22 605 33 15
Appendix E: Warranty 391 Pre-Installation Manual
E-21
Applied Biosystems
Region
Telephone
Dial...
Fax
Dial...
Russia (Moskva)
7 095 935 8888
7 095 564 8787
South East Europe (Zagreb, Croatia)
385 1 34 91 927
385 1 34 91 840
Spain (Tres Cantos)
34 (0)91 806 1210
34 (0)91 806 1206
Sweden (Stockholm)
46 (0)8 619 4400
46 (0)8 619 4401
Switzerland (Rotkreuz)
41 (0)41 799 7777
41 (0)41 790 0676
The Netherlands (Nieuwerkerk a/d IJssel)
31 (0)180 331400
31 (0)180 331409
United Kingdom (Warrington, Cheshire)
44 (0)1925 825650
44 (0)1925 282502
All other countries not listed
(Warrington, UK)
44 (0)1925 282481
44 (0)1925 282509
81 3 5566 6230
81 3 5566 6507
Japan
Japan (Hacchobori, Chuo-Ku, Tokyo)
Latin America
Del.A. Obregon, Mexico
305-670-4350
305-670-4349
To Reach Technical Support Through the Internet
We strongly encourage you to visit our Web site for answers to frequently asked questions and for
more information about our products. You can also order technical documents or an index of available documents and have them faxed or e-mailed to you through our site. The Applied Biosystems
Web site address is
http://www.appliedbiosystems.com/techsupp
To submit technical questions from North America or Europe:
Step
Action
5
Access the Applied Biosystems Technical Support Web site.
6
Under the Troubleshooting heading, click Support Request Forms, then select the relevant
support region for the product area of interest.
7
Enter the requested information and your question in the displayed form, then click Ask Us RIGHT
NOW (blue button with yellow text).
8
Enter the required information in the next form (if you have not already done so), then click Ask
Us RIGHT NOW.
You will receive an e-mail reply to your question from one of our technical experts within 24 to 48
hours.
E-22
Appendix E: Warranty 391 Pre-Installation Manual
Applied Biosystems
To Obtain Documents on Demand
Free, 24-hour access to Applied Biosystems technical documents, including MSDSs, is available
by fax or e-mail or by download from our Web site.
To order
documents...
Then...
by index number
a. Access the Applied Biosystems Technical Support Web site at
http://www.appliedbiosystems.com/techsupp
b. Click the Index link for the document type you want, then find the document you
want and record the index number.
c. Use the index number when requesting documents following the procedures
below.
by phone for fax
delivery
a. From the U.S. or Canada, call 1-800-487-6809, or
from outside the U.S. and Canada, call 1-858-712-0317.
b. Follow the voice instructions to order the documents you want.
Note
through the
Internet for fax or
e-mail delivery
There is a limit of five documents per request.
a. Access the Applied Biosystems Technical Support Web site at
http://www.appliedbiosystems.com/techsupp
b. Under Resource Libraries, click the type of document you want.
c. Enter or select the requested information in the displayed form, then click Search.
d. In the displayed search results, select a check box for the method of delivery for
each document that matches your criteria, then click Deliver Selected Documents
Now (or click the PDF icon for the document to download it immediately).
e. Fill in the information form (if you have not previously done so), then click Deliver
Selected Documents Now to submit your order.
Note There is a limit of five documents per request for fax delivery but no limit on
the number of documents you can order for e-mail delivery.
Appendix E: Warranty 391 Pre-Installation Manual
E-23
Applied Biosystems
AB LIMITED WARRANTY
Applied Biosystems (“AB”) warrants to the Customer that, for a period ending on the earlier of one
year from completion of installation or 15 months from the date of shipment to the Customer (the
“Warranty Period”), the AB Model 391 purchased by the customer (the “Instrument”) will be free
from defects in material and workmanship, and will perform in accordance with the performance
specifications contained in the applicable AB product literature (the “Specifications”).
This Warranty does not apply to the Instrument’s valves, reagent lines, or performance, unless the
Customer uses only reagents and solvents supplied by AB or expressly recommended by AB, or to
any damages caused by reagents or solvents not supplied by AB, even though recommended by AB.
This Warranty does not extend to any Instrument or part thereof that has been subjected to misuse,
neglect or accident, that has been modified or repaired by anyone other than AB or that has not been
used in accordance with the instructions contained in the Instrument Operator’s Manual. Nor does
this Warranty cover any Customer-installable consumable parts for the Instrument that are listed in
the Instrument Operator’s Manual.
AB’s obligation under this warranty is limited to repairs or replacements that AB deems necessary
to correct covered defects or failures of which AB is notified prior to expiration of the Warranty
Period. All repairs and replacements under this Warranty shall be performed by AB onsite at the
Customer’s location at AB’s expense.
No agent, employee, or representative of AB has any authority to bind AB to any affirmation, representation, or warranty concerning the Instrument that is not contained in the printed product literature or this Warranty Statement. Any such affirmation, representation or warranty made by any
agent, employee, or representative of AB shall not be binding on AB.
AB shall not be liable for any incidental, special, or consequential loss, damage or expense directly
or indirectly arising from the use of the Instrument. AB makes no warranty whatsoever in regard to
products or parts furnished by third parties; such products or parts will be subject to the warranties,
if any, of their respective manufacturers.
This Warranty is limited to the original Customer and is not transferable.
THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS EXPRESSLY IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPLIED WARRANTY OF
MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND OF ANY OTHER
OBLIGATION ON THE PART OF AB.
E-24
Appendix E: Warranty 391 Pre-Installation Manual
Headquarters
850 Lincoln Centre Drive
Foster City, CA 94404 USA
Phone: +1 650.638.5800
Toll Free: +1 800.345.5224
Fax: +1 650.638.5884
Worldwide Sales Offices
Applied Biosystems vast distribution and
service network, composed of highly trained
support and applications personnel, reaches
into 150 countries on six continents. For
international office locations, please call our
local office or refer to our web site at
www.appliedbiosystems.com.
www.appliedbiosystems.com
Applera Corporation is committed to providing
the world’s leading technology and information
for life scientists. Applera Corporation consists of
the Applied Biosystems and Celera Genomics
businesses.
Printed in the USA, 06/2002
Part Number 900937 Rev. D
an Applera business