Thermo Fisher Scientific TaqMan® Universal PCR Master Mix User guide

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

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.

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.

Contents

3

TaqMan® Universal PCR Master Mix User Guide

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

Rn and ΔRn 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

4TaqMan® Universal PCR Master Mix User Guide

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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

If the gene does not contain introns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Probe and primer design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

General probe design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

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TaqMan® Universal PCR Master Mix User Guide

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General primer design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Calculation of primer and probe concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Calculate oligonucleotide concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

An example calculation of primer concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

An 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

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Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Limited Product Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

7

TaqMan® Universal PCR Master Mix User Guide

About this guide

Revision history

Purpose of the guide

This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)assays.

Because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.Fordetailedinformation

aboutdataanalysisortheproceduresoutlinedinthisprotocol,consulttheappropriate

documentationforyourinstrument.

User attention words

Two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product.

IMPORTANT! Providesinformationthatisnecessaryforproperinstrumentoperation

oraccuratechemistrykituse.

Revision Date Description

E March 2014 Update to genotyping content to include:

•TaqMan

® SNP Genotyping PCR

•TaqMan

® Drug Metabolism PCR

Updated to current style and format.

D July 2010

8TaqMan® Universal PCR Master Mix User Guide

About this guide

User attention words

1

9

TaqMan® Universal PCR Master Mix User Guide

Product Information

■Productdescription....................................................9

■Kitcontentsandstorage ...............................................10

■Compatiblereal‐timeinstruments .......................................10

■Materialsrequiredbutnotincluded.....................................11

Product description

TaqMan®UniversalPCRMasterMixisaconvenientmixofcomponents(except

primers,probes,template,andwater)necessarytoperformareal‐timepolymerase

chainreaction(PCR).TaqMan®UniversalPCRMasterMixcanbeusedtoamplify

knownsequencesofgenomic,plasmid,orcomplementaryDNA(cDNA)targetsfora

varietyofapplications,includingquantitationandgenotyping.Themixisavailable

withorwithoutAmpErase®UNG.

ThisdocumentdescribestheuseoftheTaqMan®UniversalPCRMasterMixwith

pre‐optimized:

• TaqMan®GeneExpressionAssays

• TaqMan®MicroRNAAssays

• TaqMan®DrugMetabolismGenotypingAssays

• TaqMan®SNPGenotypingAssays

•CustomTaqMan®ProbesandSequenceDetectionPrimers

toamplifyyourDNAtargetofchoice.Guidelinesarealsoprovidedforthedesignand

optimizationofcustomprimersandTaqMan®probes.

InRNAquantitationassaystheTaqMan®UniversalPCRMasterMixisusedinthe

secondstepofa2‐stepreversetranscription–polymerasechainreaction(RT‐PCR)

protocol.ThecDNAtemplateusedwiththemastermixcanbegeneratedinareverse

transcriptionreactionusingkitsavailablefromLifeTechnologies.

10 TaqMan® Universal PCR Master Mix User Guide

Chapter1 Product Information

Kit contents and storage

1

Kit contents and storage

TheTaqMan®UniversalPCRMasterMixissuppliedat2Xconcentrationandcontains:

•AmpliTaqGold®DNAPolymerase,UP(UltraPure)

•Uracil‐Nglycosylase(UNG)

•dNTPswithdUTP

•ROX

™PassiveReference

•Optimizedbuffercomponents

Note: TaqMan®UniversalPCRMasterMix,noAmpErase®UNG,containsallthe

aboveingredientsexceptUNG.

TaqMan®UniversalPCRMasterMix,withornoAmpErase®UNG,issuppliedat2X

concentrationandisavailableinthefollowingvolumes:

Compatible real-time instruments

TheTaqMan®UniversalPCRMasterMix,withorwithoutAmpErase®UNG,maybe

usedforreal‐timeorplateread(endpoint)detectionofDNAorcDNA.Analysisis

performedusinganyofthefollowingreal‐timePCRsystemsavailablefromLife

Technologies:

•StepOne

™orStepOnePlus™Real‐TimePCRSystem

•AppliedBiosystems®7300/7500/7500FastReal‐TimePCRSystem

•AppliedBiosystems®7900HT/7900HTFastReal‐TimePCRSystem

•AppliedBiosystems®ViiA™7Real‐TimePCRSystem

• QuantStudio™6FlexReal‐TimePCRSystem

• QuantStudio™7FlexReal‐TimePCRSystem

• QuantStudio™12kFlexSystem

Master Mix Catalog Number Quantity

# of reactions

Storage

20–µL 50-µL

TaqMan® Universal PCR Master Mix 4304437, 1–Pack 1 × 5 mL 500 200 2°C to 8°C.

4364338, 2–Pack 2 × 5 mL 1000 400

4364340, 5–Pack 5 × 5 mL 2500 1000

4305719, 10–Pack 10 × 5 mL 5000 2000

4318157, 10 Unit Pack 10 × 5 mL 5000 2000

4326708, 1 Bulk Pack 1 × 50 mL 5000 2000

TaqMan® Universal PCR Master Mix,

No AmpErase® UNG

4324018, 1–Pack 1 × 5 mL 500 200 2°C to 8°C.

4364341, 2–Pack 2 × 5 mL 1000 400

4364343, 5–Pack 5 × 5 mL 2500 1000

4324020, 10 Unit Pack 10 × 5 mL 5000 2000

4326614, 1 Bulk Pack 1 × 50 mL 5000 2000

11

TaqMan® Universal PCR Master Mix User Guide

Chapter1 Product Information

Materials required but not included 1

Fast instruments

and thermal

cycling conditions

IMPORTANT! UseTaqMan®UniversalPCRMasterMixwithStandardmodethermal

cyclingconditionsonly.TaqMan®UniversalPCRMasterMixisnotsupportedforuse

withFastModethermalcyclingconditions.WhenusingTaqMan®UniversalPCR

MasterMixontheStepOne™,StepOnePlus™,7500Fast,7900HTFast,ViiA7Fast96‐

well,orQuantStudioFast96‐wellinstruments,useStandardmodethermalcycling

conditions.IfyouuseassaysotherthantheTaqMan®assays,orusethermalcycling

conditionsotherthanthosespecifiedinthisprotocol,validateyourassaysandre‐

optimizeyourthermalcyclingconditionsasneeded.

Materials required but not included

Beforehandlinganychemicals,refertotheSDSprovidedbythemanufacturer,and

observeallrelevantprecautions.

Materials Catalog no.

High Capacity RNA-to-cDNA™ Kit, 50 rxns 4387406

SuperScript® VILO™ cDNA

Synthesis Kit

50 reactions 11754050

250 reactions 11754250

RNase inhibitor N8080119

TaqMan® Gene Expression Assays, inventoried 4331182

TaqMan® Gene Expression Assays, made-to-order 4351372

Custom TaqMan® Gene

Expression Assays

Small-scale (20X, 144 × 50-µL rxns) 4331348

Medium-scale (20X, 300 × 50-µL rxns) 4332078

Large-scale (60X, 1160 × 50-µL rxns) 4332079

TaqMan® SNP Genotyping

Assays

Small-scale (40X, 300 × 25-µL rxns) 4351379

Custom TaqMan® SNP

Genotyping Assays

Small-scale (40X, 300 × 25-µL rxns) 4331349

TaqMan® Drug Metabolism

Genotyping Assays

Small-scale (20X, 150 × 25-µL rxns) 4362691

TaqMan® PreAmp Master Mix Kit, 40 rxns 4384267

Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0,

made using DNase-free, RNase-free sterile-filtered water)

AM9849

DNAZap™ Solution, two, 250-mL bottles AM9890

RT-PCR Grade Water, ten, 1.75-mL bottles AM9935

DNase-free water AM9914G

12 TaqMan® Universal PCR Master Mix User Guide

Chapter1 Product Information

Materials required but not included

1

2

13

TaqMan® Universal PCR Master Mix User Guide

Gene expression quantitation

Thischapterprovidesaprotocolforperforming2‐stepreversetranscription

polymerasechainreaction(RT‐PCR)usingTaqMan®UniversalPCRMasterMixwith

TaqMan®GeneExpressionAssays,CustomTaqMan®GeneExpressionAssays,or

CustomTaqMan®ProbesandSequenceDetectionPrimers.

■Reversetranscription ..................................................13

■Real‐timePCRamplification............................................14

■Setupplatedocumentorexperiment ....................................15

■PrepareandrunthePCRreactionplate..................................16

Reverse transcription

Synthesisofsingle‐strandedcDNAfromRNAisthefirststepinthetwo‐stepRT‐PCR

process,whichrequiresyouto:

1. Preparethereversetranscription(RT)reactionmix.

2. PreparetheRTreactionplate.

3. Performreversetranscription.

ToobtaincDNAfromRNAsampleswerecommendusingaLifeTechnologiesreverse

transcriptionkit.

ForadditionalRTguidelinesandinstructions,refertotheappropriateprotocol.You

candownloadtheprotocolsforLifeTechnologieskitsat:www.lifetechnologies.com.

Note: Foradditionaldocumentation,see“Obtainingsupport”onpage70.

RNA template

guidelines

ThequalityofyourresultsisdirectlyrelatedtothepurityofyourRNAtemplate.For

optimalperformanceuseRNAthatis:

•FreeofinhibitorsofreversetranscriptionandPCR.

• DissolvedinTEbufferorPCR‐compatiblebuffer.

•FreeofRNaseactivity.

•FreeofgenomicDNAcontamination.

Reagent and

sample

preparation

guidelines

Toensureoptimalperformance:

•Usenuclease‐freepipettipsandreagentstominimizedegradationoftheRNA.

•ObservestandardlaboratorypracticeswhenhandlingRNA.

14 TaqMan® Universal PCR Master Mix User Guide

Chapter2 Gene expression quantitation

Real-time PCR amplification

2

Real-time PCR amplification

TargetamplificationusingcDNAasthetemplateisthesecondstepintheRT‐PCR

process.Inthisstep,theDNApolymerase(fromtheTaqMan®UniversalPCRMaster

Mix)amplifiestargetcDNAsynthesizedfromtheRNAsample,usingsequence‐

specificprimersandaTaqMan®probe(forexample,aprobefromtheTaqMan®Gene

ExpressionAssaymix).

IMPORTANT! YoumustperformthePCRsteponareal‐timePCRsystem.Traditional

thermalcyclerscannotbeusedbecausetheycannotdetectandrecordthefluorescent

signalsgeneratedbythecleavageofTaqMan®probes.

Note: IfyouchoosetouseCustomTaqMan®ProbesandSequenceDetectionPrimers,

ratherthanaTaqMan®GeneExpressionAssayoraCustomTaqMan®Gene

ExpressionAssay,see“CustomTaqMan®assaydesign”onpage 47formore

information.

PCR reagent

handling and

preparation

FollowingtheseguidelinesensuresoptimalPCRperformance:

•KeeptheTaqMan®assaysinthefreezer,awayfromlight,untilyouarereadyto

usethem.Excessiveexposuretolightmayaffectthefluorescentprobes.

•Priortouse:

–MixtheTaqMan®UniversalPCRMasterMixthoroughlybyswirlingthe

bottle.

–ThawfrozenTaqMan®assaysbyplacingthemonice.Whenthawed,

resuspendthesamplesbyvortexing,thenbrieflycentrifugetocollect.

–Thawfrozensamplesbyplacingthemonice.Whenthawed,resuspendthe

samplesbybrieflyvortexingandthencentrifugethetubestocollect.

Determine the

number of required

reactions

Determinethenumberofreactionstoperformforeachassay.Werecommend

performingfourreplicatesofeachreaction.Include10%extravolumetocompensate

forthevolumelossthatoccursduringpipetting.Forexample,ifusinga96‐wellplate,

prepareenoughreactionmixforapproximately106 reactions.

Besuretoincludeoneachplate:

•AgeneexpressionassayforeachcDNAsample.

• Endogenouscontrolassays.

•Notemplatecontrols(NTCs)foreachassayontheplate.

IMPORTANT! Youcanrunmultipleassaysononereactionplate.Includecontrolsfor

eachassaythatyourunonaplate.

15

TaqMan® Universal PCR Master Mix User Guide

Chapter2 Gene expression quantitation

Real-time PCR amplification 2

Set up plate

document or

experiment

Refertoyourinstrumentuserguideforinstructionsonhowtoconfiguretheplate

documentorexperiment.See“Obtainingsupport”onpage 70toobtainuser

documentationforLifeTechnologiesreal‐timePCRsystems.

Whencreatingplatedocuments/experiments,usethefollowingparameters:

•ThermalCyclingParameters:

IMPORTANT! Omitthe2‐minute,50°CstepifyouareusingTaqMan®Universal

PCRMasterMix,noAmpErase®UNG.

•RunMode:Standardmode

•SampleVolume:

•AutoIncrementSettings:Acceptdefaultvalues(defaultis0)

• DataCollection:Acceptdefaultvalues(defaultis60°C)

• RampRateSettings:Acceptdefaultvalues(defaultisStandard)

IMPORTANT! TaqMan®UniversalPCRMasterMixisnotsupportedforusewithFast

Modethermalcyclingconditions.WhenusingTaqMan®UniversalPCRMasterMixon

theStepOne™,StepOnePlus™,7500Fast,7900HTFast,ViiA7Fast96‐well,or

QuantStudioFast96‐wellinstruments,useStandardmodethermalcyclingconditions.

Parameter UNG

incubation†

† Required for optimal UNG activity. If using TaqMan® Universal PCR Master Mix, no AmpErase®

UNG, this step is not necessary.

Polymerase

activation‡

‡ Required to activate the DNA Polymerase.

PCR

(40 cycles)

Hold Hold Denature Anneal/extend

Temperature 50°C 95°C 95°C 60°C

Time (mm:ss) 02:00 10:00 00:15 01:00

Plate format Reaction volume (µL)

MicroAmp® Optical 96-Well Reaction Plate 20–50

MicroAmp® Optical 384-Well Reaction Plate 10–20

16 TaqMan® Universal PCR Master Mix User Guide

Chapter2 Gene expression quantitation

Real-time PCR amplification

2

Prepare and run

the PCR reaction

plate

1. Preparethereactionmixforeachsampleusingthecomponentslistedbelow.

•CalculatethevolumeofeachcomponentofthePCRreactionmixby

multiplyingthevolumeofeachcomponentbythenumberofreplicatesfor

eachsample.

•Werecommendperformingfourtechnicalreplicatesofeachreaction.Select

thereactionsizedependingonthereactionplateused.Prepare110%ofthe

requiredvolumetoaccountforpipettingerror.

IMPORTANT! ForoptimalperformanceofTaqMan®GeneExpressionAssays,use

1–100 ngofcDNAper20‐or50‐μL reaction.

2. Capthetube(s)andvortexbrieflytomixthesolutions.

3. Centrifugethetube(s)brieflytospindownthecontentsandeliminateanyair

bubblesfromthesolutions.

4. Transfertheappropriatevolumeofeachreactionmixturetoeachwellofan

opticalreactionplate.

5. CovertheplatewithaMicroAmp®OpticalAdhesiveFilm.Forstandard96‐well

plates,youmayuseMicroAmp®OpticalCaps.

6. Centrifugetheplatebrieflytospindownthecontentsandeliminateairbubbles

fromthesolutions.

7. Applyacompressionpadtotheplateifrequiredbyyourreal‐timePCRsystem.

8. Inthesystemsoftware,opentheplatedocumentorexperimentthatcorresponds

tothereactionplate.

9. Loadthereactionplateintothereal‐timePCRsystem.

10. Starttherun.

Component

Volume (µL)

per reaction

Final conc.

50-µL

rxns.

20-µL

rxns.

TaqMan® Universal PCR Master Mix (2X) 25.0 10.0 1X

TaqMan® Gene Expression Assay (20X)†

†See www.LifeTechnologies.com for TaqMan® Gene Expression Assay information.

2.5 1.0 1X

cDNA template + H2O‡

‡ Use 1–100 ng of cDNA plus RNase-free water.

22.5 9.0 1–100 ng

Total Volume 50.0 20.0 —

3

17

TaqMan® Universal PCR Master Mix User Guide

Data Analysis

Thischapterprovidesgeneralguidelinesforanalyzingdataobtainedfromgene

expressionassays.

■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

Overview

Dataanalysisvariesdependingontheproduct,assayandreal‐timePCRsystemused.

RefertotheappropriatePCRinstrumentuserguidefordetailedanalysisinstructions.

See“DocumentationandSupport”onpage69formoreinformation.

Thegeneralprocessforanalyzinggeneexpressiondatainvolves:

1. ViewingtheamplificationplotsfortheentirePCRreactionplate.

2. Settingthebaselineandthresholdvaluestodeterminethethresholdcycles(CT)

fortheamplificationcurves.

3. UsingtherelativestandardcurvemethodorthecomparativeCTmethodto

analyzethedata.

Baseline and

threshold values

Whenusingareal‐timePCRsystem,youcanusethesoftwaretosetthebaselineand

thresholdfortheamplificationcurveseitherautomaticallyormanually.

•ThebaselinereferstotheinitialcyclesofPCRinwhichthereisslightchangein

fluorescencesignal.

•TheintersectionofthethresholdwiththeamplificationplotdefinestheCTinreal‐

timePCRassays.Thethresholdissetabovethebackgroundandwithinthe

exponentialgrowthphaseoftheamplificationcurve.

18 TaqMan® Universal PCR Master Mix User Guide

Chapter3 Data Analysis

Overview

3

Normalization ThePassiveReferenceisadyeincludedintheTaqMan®UniversalPCRMasterMix

anddoesnotparticipateinthe5´nucleasePCR.ThePassiveReferenceprovidesan

internalreferencetowhichthereporter‐dyesignalcanbenormalizedduringdata

analysis.Normalizationisnecessarytocorrectforfluorescentfluctuationsdueto

changesinconcentrationorvolume.

Rn and ΔRn values Normalizationisaccomplishedbydividingtheemissionintensityofthereporterdye

bytheemissionintensityofthePassiveReferencetoobtainaratiodefinedastheRn

(normalizedreporter)foragivenreactiontube.

Rn+istheRnvalueofareactioncontainingallcomponentsincludingthetemplate.

Rn–istheRnvalueofanunreactedsample.Thisvaluemaybeobtainedfromtheearly

cyclesofareal‐timerun,thosecyclespriortoadetectableincreaseinfluorescence.

Thisvaluemayalsobeobtainedfromareactionnotcontainingtemplate.

ΔRnisthedifferencebetweentheRn+valueandtheRn–value.Itreliablyindicatesthe

magnitudeofthesignalgeneratedbythegivensetofPCRconditions.

Thefollowingequationexpressestherelationshipoftheseterms:

ΔRn=(Rn+)–(Rn–)

where:

Automatic

calculation of the

baseline and

threshold

Thesystemsoftwarecalculatesbaselineandthresholdvaluesforadetectorbasedon

theassumptionthatthedataexhibitthe“typical”amplificationcurveshownbelow.

Experimentalerror(suchascontaminationorpipettingerrors)canproduceatypical

datathatcancausethesoftwarealgorithmtogenerateincorrectbaselineandthreshold

valuesfortheassociateddetector.

IMPORTANT!Afterananalysis,verifythatthebaselineandthresholdwerecalled

correctlyforeachwellbyviewingtheresultingamplificationplots,andadjustthe

valuesmanuallyifnecessary.

Rn+ = Emission Intensity of Reporter PCR with template

Emission Intensity of Passive Reference

Rn– = Emission Intensity of Reporter PCR without template or early

cycles of a real-time reaction

Emission Intensity of Passive Reference

a

Rn

a. Plateau phase

b. Linear phase

c. Exponential

(geometric)

phase

d. Background

e. Baseline

b

c

d

e

Threshold

19

TaqMan® Universal PCR Master Mix User Guide

Chapter3 Data Analysis

Overview 3

Manual setting of

the baseline and

threshold

Ifyouusethesystemsoftwaretosetthebaselineandthresholdvaluesmanuallyfor

anydetector/assayinthestudy,performanadjustmentprocedureforeachdetector/

assay.Refertoyourreal‐timePCRsystemdocumentationforguidanceonmanually

settingandadjustingyourthresholdandbaseline.

Correct and incorrect threshold settings

Threshold set correctly

The threshold is set in the exponential phase of the

amplification curve. Threshold settings above or below

the optimum increase the standard deviation of the

replicate groups.

Threshold set too low

The threshold is set below the exponential phase of the

amplification curve. The standard deviation is

significantly higher than that for a plot where the

threshold is set correctly. Set the threshold up into the

exponential phase of the curve.

Threshold set too high

The threshold is set above the exponential phase of the

amplification curve. The standard deviation is

significantly higher than that for a plot where the

threshold is set correctly. Set the threshold down into

the exponential phase of the curve.

20 TaqMan® Universal PCR Master Mix User Guide

Chapter3 Data Analysis

Overview

3

Analyze the data YoucanperformtwotypesofquantitationusingtheTaqMan®UniversalPCRMaster

Mix:

•Relativequantitationcomparestherelativeexpressionofatargetgenebetween

anunknownandareferencesample.Youcanperformrelativequantitationusing

thestandardcurvemethodorthecomparativeCTmethod.

•AbsolutequantitationcomparestheCTofanunknownsampleagainstastandard

curvewithknowncopynumbers.

Quantitation of

cDNA relative to a

calibrator sample

Geneexpressioncanbemeasuredbycomparingtherelativeexpressionofatarget

geneinaunknownsampleandinaphysiologicalreferencesample.Inatypical

experiment,geneexpressionlevelsarestudiedasafunctionofeitheratreatmentof

cellsinculture,ofpatients,oroftissuetype.Thecalibratorsampleineachcaseisthe

cDNAfromeithertheuntreatedcellsorpatients,oraspecifictissuetype.

AllquantitationsarealsonormalizedtoanendogenouscontrolsuchasGAPDHto

accountforvariabilityintheinitialconcentrationandqualityofthetotalRNAandin

theconversionefficiencyofthereversetranscriptionreaction.Allampliconsinthese

determinationsshouldfollowthePrimerExpress®Softwareamplicondesigncriteria.

Correct and incorrect baseline settings

Baseline set correctly

The amplification curve begins after the maximum

baseline.

Baseline set too low

The amplification curve begins too far to the right of the

maximum baseline. Increase the End Cycle value.

Baseline set too high

The amplification curve begins before the maximum

baseline. Decrease the End Cycle value.

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Thermo Fisher Scientific TaqMan® Universal PCR Master Mix User guide

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