TaqMan Universal PCR Master Mix - Thermo Fisher Scientific

1 USER GUIDEFor Research Use Only. Not for use in diagnostic Universal PCR Master MixCatalog Numbers4324018, 4364341, 4364343, 4324020, 4326614, 4304437, 4364338, 4364340, 4305719, 4318157, 4326708 Publication Number4304449 RevisionEFor Research Use Only. Not for use in diagnostic information in this guide is subject to change without 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 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.

2 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 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.

3 TRI Reagent is a registered trademark of Molecular Research Center, Inc. Pipetman is a registered trademark of Gilson Eppendorf is a registered tradmark of Eppendorf AG. 2014 Life Technologies Corporation. All rights q M a n Universal PCR Master Mix User GuideAbout 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.

4 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 .. 18Rn 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 .. 214Ta q M a n Universal PCR Master Mix User GuideContentsDetermine the number of required reactions .. 21 Perform genotyping.

5 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.

6 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 .. 46 About ROX passive reference .. 46 APPENDIX D Custom TaqMan assay design .. 47 Before you begin .. 47 Identifying target sequence and amplicon size .. 47 Target template .. 47 Amplicon .. 47 General amplicon site selection guidelines .. 47If the gene does not contain introns .. 48 Probe and primer design .. 48 General probe design guidelines .. 485Ta q M a n Universal PCR Master Mix User GuideContentsGeneral primer design guidelines .. 48 Calculation of primer and probe concentrations.

7 49 Calculate oligonucleotide concentrations .. 49An example calculation of primer concentration .. 49An example calculation of probe concentration .. 50 Determine optimal primer concentrations .. 50 Primer concentrations to test .. 50 Prepare and run the real-time qPCRs .. 51 Determine optimal probe concentration .. 52 Probe concentrations to test .. 52 Prepare and run the real-time qPCRs .. 52 Evaluate the results .. 53 Performing routine analysis .. 53 APPENDIX E Allelic discrimination probe/primer design .. 55 Before you begin .. 55 Identifying target sequence and amplicon size .. 55 Target template defined .. 55 General amplicon site selection guidelines .. 55 Probe and primer design .. 56 General probe design guidelines .. 56 General primer design guidelines.

8 56 Calculation of primer and probe concentrations .. 57 Determine optimal primer concentrations .. 57 Primer concentrations to test .. 57 Prepare and run the real-time qPCRs .. 57 Determine optimal probe concentrations .. 58 Probe concentration considerations .. 58 Performing routine analysis .. 58 Analyze the results .. 59 APPENDIX F Ordering Information .. 61 How to order .. 61 Real-time PCR systems, PCR systems, and consumables .. 62 Gene expression assay and array products .. 64 Reverse transcription kits and reagents .. 65 Optional user-supplied reagents for gene expression quantitation .. 65 Consumables and equipment .. 66 APPENDIX G Safety .. 67 Chemical safety .. 67 Biological hazard safety .. 686Ta q M a n Universal PCR Master Mix User GuideContentsDocumentation and Support.

9 69 Related documentation .. 69 Obtaining SDSs .. 69 Obtaining Certificates of Analysis .. 69 Obtaining support .. 70 Limited Product Warranty .. 707Ta q M a n Universal PCR Master Mix User GuideAbout this guideRevision historyPurpose of the guideThis user guide describes the two primary applications of the TaqMan Universal PCR Master Mix: quantitative RT PCR and genotyping. Although TaqMan Universal PCR Master Mix can be used in a broad variety of PCR applications, this document primarily describes the use of the Master mix with pre optimized TaqMan assays. General guidelines are provided for the design and optimization of custom quantitation and allelic discrimination (genotyping) analysis methods vary greatly between applications, this user guide provides general guidelines for the analysis of data generated from experiments that use TaqMan Universal PCR Master Mix and TaqMan assays.

10 For detailed information about data analysis or the procedures outlined in this protocol, consult the appropriate documentation for your attention wordsTwo user attention words may appear in this document. Each word implies a particular level of observation or action as described below:Note:Provides information that may be of interest or help but is not critical to the use of the !Provides information that is necessary for proper instrument operation or accurate chemistry kit 2014 Update to genotyping content to include: TaqMan SNP Genotyping PCR TaqMan Drug Metabolism PCRU pdated to current style and 20108Ta q M a n Universal PCR Master Mix User GuideAbout this guide User attention words 19Ta q M a n Universal PCR Master Mix User Guide Product Information Product description.