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Taqman® Universal PCR Master Mix User Guide 3 Contents USER GUIDE TaqMan® Universal PCR Master Mix Catalog Numbers 4324018, 4364341, 4364343, 4324020, 4326614, 4304437, 4364338, 4364340, 4305719, 4318157, 4326708 Publication Number 4304449 Revision E For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures. The information in this guide is subject to change without notice. Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses. DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. TaqMan, AmpliTaq Gold, and AmpErase are registered trademarks of Roche Molecular Systems, Inc., TaqMan used under permission and license. Microsoft and Excel are registered trademarks of Microsoft Corporation. Adobe and Reader are registered trademarks of Adobe Systems, Inc. TRI Reagent is a registered trademark of Molecular Research Center, Inc. Pipetman is a registered trademark of Gilson S.A.S. Eppendorf is a registered tradmark of Eppendorf AG. © 2014 Life Technologies Corporation. All rights reserved. Contents About this guide . 7 Revision history . 7 Purpose of the guide . 7 User attention words . 7 ■ CHAPTER 1 Product Information . 9 Product description . 9 Kit contents and storage . 10 Compatible real-time instruments . 10 Fast instruments and thermal cycling conditions . 11 Materials required but not included . 11 ■ CHAPTER 2 Gene expression quantitation . 13 Reverse transcription . 13 RNA template guidelines . 13 Reagent and sample preparation guidelines . 13 Real-time PCR amplification . 14 PCR reagent handling and preparation . 14 Determine the number of required reactions . 14 Set up plate document or experiment . 15 Prepare and run the PCR reaction plate . 16 ■ CHAPTER 3 Data Analysis . 17 Overview . 17 Baseline and threshold values . 17 Normalization . 18 Δ Rn and Rn values . 18 Automatic calculation of the baseline and threshold . 18 Manual setting of the baseline and threshold . 19 Analyze the data . 20 Quantitation of cDNA relative to a calibrator sample . 20 ■ CHAPTER 4 Genotyping . 21 Before you begin . 21 Quantitate the DNA . 21 TaqMan® Universal PCR Master Mix User Guide 3 Contents Determine the number of required reactions . 21 Perform genotyping . 22 Prepare the PCR reaction mix . 22 Perform TaqMan® SNP Genotyping PCR . 23 Perform TaqMan® Drug Metabolism Genotyping PCR . 24 Read the plate . 24 Analyze the results . 25 ■ APPENDIX A Troubleshooting . 27 Troubleshooting gene expression experiments . 28 Troubleshooting genotyping experiments . 35 Observation 1: No or low amplification . 35 Observation 2: No clusters . 36 Observation 3: Clusters too close . 36 Observation 4: Too many clusters . 37 Observation 5: “Chicken-feet” clusters . 38 ■ APPENDIX B PCR Good Laboratory Practices . 39 Sample preparation . 39 Preventing contamination . 40 False positives . 40 Uracil-N glycosylase (UNG) . 40 Prevention of PCR product carryover . 41 Fluorescent contaminants . 41 ■ APPENDIX C Chemistry Overview . 43 About two-step RT-PCR . 43 Basics of the 5’ nuclease assay . 44 About TaqMan® MGB Probes . 46 About AmpliTaq Gold® DNA Polymerase, (UP) Ultra Pure . 46 About uracil-N glycosylase . ..
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