GenomeLab GeXP - SCIEX

1 A53995 ACDecember 2009 Beckman Coulter, S. Kraemer Blvd., Brea, CA 92821 Copyright 2009 Beckman Coulter, GeXPTroubleshooting GuideiiCopyright, Licenses and TrademarksCopyright Beckman Coulter, Inc., 2009. All rights reserved. No part of this publication may be reproduced, transcribed, transmitted, or translated into any language in any form by any means without the written permission of Beckman Coulter, and TrademarksBeckman Coulter is a registered trademark of Beckman Coulter, is a trademark of Beckman Coulter, other trademarks and registered trademarks are property of their respective GuidePN A53995AC iiiSection 1 Introduction.

2 1 Section 2 General Diagnostic Guidelines .. Evaluating the Raw Data .. 4 Examining Signal Strength and Multiplex Profile ..4 Checking the Raw Data.. Evaluating the Current Profiles .. 6 Confirming the Separation Current ..6 Causes of Current Abnormalities ..8 Section 3 Instrumentation & Chemistry .. Performing Instrument Diagnostics .. 9 Running Size Standard-400 ..9 Interpreting the Results ..10 Running the Sequencing Test Sample ..10 Interpreting the Results .. Testing the Chemistry .. 11 Testing the gexp Start Kit and Experimental Process ..11 Interpreting the Results ..12 Examining the Experimental Process.

3 Testing Third-Party Products .. 12 Interpreting the Results ..13 Testing the Experimental RNA and Custom Multiplex Primer ..13 Interpreting the Results ..14 Section 4 Reference Tables .. Multiplex Primer Design .. gexp Chemistry .. gexp Separation and Fragment Analysis .. eXpress Profiler and Analysis .. 28 Section 5 Questions & Answers .. eXpress Profiler.. Multiplex Primer Design .. gexp Chemistry and Fragment Analysis .. eXpress Analysis .. 34 Section 6 Appendix A .. 37 Troubleshooting How Tos 37 Troubleshooting Multiplex Design with Verbose Mode ..37 Repairing a Corrupt TDF File.

4 37 Creating a Multiplex without an Internet Connection ..38 Designing Primers to Detect Alternative Transcripts ..38 Modifying a Previously Designed Multiplex ..39 Table of ContentsGenomeLab GeXPPN A53995AC iv Importing Two or More Plates into eXpress Analyzer ..39 Section 7 Appendix B .. 41 Reference Figures 41 Troubleshooting GuidePN A53995AC 1 IntroductionOverview1 IntroductionOverviewThe gexp Troubleshooting guide has been designed to help you identify and correct the issues most commonly encountered with the GenomeLab gexp Genetic Analysis System. Read through the entire gexp Troubleshooting Guide first, before performing troubleshooting experiments.

5 Begin with General Diagnostics, which will inform you on the data you will need and where it can be found. Next, Instrumentation & Chemistry will provide a systematic approach to enable you to effectively diagnose the problem. See Figure on page 2 for the gexp Troubleshooting Workflow. This serves as a visual aide for the troubleshooting , the Reference Tables and Questions & Answers sections are particularly helpful for resolving specific problems. These sections can also aid in rapid identification of the most likely suspects for poor 1. 1 gexp Troubleshooting WorkflowNoNoYesYesYesNoYesNoYesView the dataIs the problem with the gexp instrument?

6 I m not sure where the problem is. Is it the problem with the chemistry?Run the Size Standard-400 Use the gexp Start Kit and control multiplex gene set kit to perform gexp reactionsAre the peak resolution and current okay?Are there current abnormalities?Do you have a KANr peak?I don t have a have KANr with all multiplex and process are resolution and current are have poor peak the Sequencing Test Sample (PN 608070)Is the current okay?Contact your Beckman Coulter Field Service RepresentativeGo to Chemistry TroubleshootingI have current protocol Test a new kitTest experimental RNA and primersDo you have a KANr peak?

7 I still don t have a KANr Troubleshooting WorkflowTest third-party productsContact your Beckman Coulter Technical Support Representative Troubleshooting GuidePN A53995AC 3 General Diagnostic GuidelinesOverview1 General Diagnostic GuidelinesOverviewThe GenomeLab gexp provides software functions that will help you to identify and resolve gexp problems. Two key features in this regard are the Raw Data and Current Profile. NOTE The Analyzed Data can also be used for troubleshooting, but only after the Raw Data and Current Profile have been shown to be the raw data, current profile and analyzed data to determine whether the issue is caused by gexp instrumentation or chemistry or possibly both.

8 Be sure to note the following aspects of each type of data for all of the affected samples and appropriate controls: Raw and Analyzed Data rBaseline for D4 (blue) and D1 (red) rSignal strength rSignal profile (level or drop-off) rLandmarks runincorporated primers (raw only) rmultiplex peaks rKANr peak rsize standard peaksSee "Evaluating the Raw Data" on page 4 for more Profile rRamping profile rMaximum separation current ( A) rLevel of current throughout separationSee "Evaluating the Current Profiles" on page 6 for more GeXPPN A53995AC 4 General Diagnostic GuidelinesEvaluating the Raw Data1. 1 Evaluating the Raw DataThe raw data generated by the gexp is displayed in the Data Monitor window of the Run Control module, during the actual separation.

9 The data is also accessible by using Fragment Analysis. Examining Signal Strength and Multiplex ProfileRefer to the signal strength and multiplex profile as the first step in diagnosing problems, when looking at raw data. rAlthough peak heights vary for different multiplex fragments, the signal strength of D4-dye (blue) labeled multiplex peaks should be fairly even across the separation after the initial unincorporated primer dye front rOne exception is the positive control, 325 nucleotide kanamycin resistance (KANr) peak, which is usually over-range in most gexp reactions rAll of the D1-dye (red) labeled size standard peaks should be appropriately spaced and approximately the same heightFigure 1.

10 1 gexp Raw DataTroubleshooting GuidePN A53995AC 5 General Diagnostic GuidelinesEvaluating the Raw DataChecking the Raw DataThe electropherogram shown in Figure shows a typical gexp separation with a start time at approximately 11 minutes for the Frag-3 separation method. The signal strength at the beginning of the separation is over-range due to excess primers remaining in the PCR reaction. This is common for gexp reactions. The multiplex fragments begin appearing at approximately minutes for this particular sample. The positive control KANr fragment appears at approximately minutes. The D1-dye (red) labeled Size Standard-400 peaks are appropriately spaced and even in data is displayed as signal intensity, measured in relative fluorescence units (RFU) vs.