Download Plexor(TM) Systems: Instrument Setup and Data Analysis for the Bio

tm270.0905.qxp
10/7/2005
9:54 AM
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Plexor™ Systems
Instrument Setup and Data Analysis
for the Bio-Rad iCycler iQ™
Real-Time PCR Detection System
All technical literature is available on the Internet at www.promega.com
Please visit the web site to verify that you are using the most current version of this Technical Manual.
I.
Description..................................................................................................................................2
II.
Plate Preparation and Amplification.....................................................................................2
III.
Instrument Setup and Thermal Cycling for qPCR and Two-Step qRT-PCR ...............3
A. Thermal Cycling Program ..............................................................................................3
B. Plate Setup.........................................................................................................................5
IV.
Instrument Setup and Thermal Cycling for One-Step qRT-PCR....................................8
A. Thermal Cycling Program ..............................................................................................8
B. Plate Setup.......................................................................................................................10
V.
Instrument Setup and Thermal Cycling for Genotyping (SNP) Assays......................12
A. Thermal Cycling Program ............................................................................................12
B. Plate Setup.......................................................................................................................14
VI.
Data Export from the iCycler iQ™
and Import into the Plexor™ Analysis Software ..................................................................17
A. Pasting Data Directly into the Plexor™ Software.....................................................17
B. Data Export and Import via Excel ...............................................................................22
VII.
Data Analysis with the Plexor™ Analysis Software........................................................28
A. Sample Definition...........................................................................................................28
B. Adjusting the Expected Target Melt Temperature ...................................................30
C. Adjusting the Y Axes of the Amplification and
Thermal Melt Curves (Optional) .................................................................................32
D. Adjusting the Baseline Region and Amplification Threshold Line (Optional) ....32
E. Generating a Standard Curve (Optional) ...................................................................33
F.
Reports .............................................................................................................................36
G. Saving and Printing the Analysis File.........................................................................38
VIII.
Troubleshooting.......................................................................................................................38
IX.
Appendix ...................................................................................................................................45
A. Plexor™ Analysis Software Operating System Compatibility ...............................45
B. Plexor™ Analysis Software Installation .....................................................................45
C. Advanced Options .........................................................................................................46
D. Manual Baseline Adjustments......................................................................................48
E. Icon Definitions ..............................................................................................................49
F.
Amplification Efficiency Calculations ........................................................................51
G. Use of an External Well Factor Plate...........................................................................51
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
9/05
Part# TM270
Page 1