SYBR Green PCR Master Mix And SYBR Green RT-PCR

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USER GUIDESYBR Green PCR Master Mix and SYBR GreenRT-PCR Reagents KitCatalog Number 4309155 (Master Mix) and 4306736 (RT-PCR Reagents Kit)Publication Part Number 4310251 Rev. GRevision Date September 2011

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.Information in this document is subject to change without notice.LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDINGBUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITSAFFILIATE(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 NOTLIMITED TO THE USE THEREOF.LIMITED USE LABEL LICENSE: Research Use OnlyThe purchase of this product conveys to the purchaser the limited, non-transferable right to use this product only to perform internal research for the solebenefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internalresearch purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services suchas reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.The purchase of this product includes a limited, non-transferable license under U.S. Patents and corresponding claims in patents and patent applicationsoutside the United States, owned by the University of Utah Research Foundation and licensed to Idaho Technology. Inc., to use only this amount of productfor dsDNA-Binding Dye assays solely for the purchaser's own internal research and development activities. No right is conveyed, expressly, by implication orestoppel, under any other patent or patent claims, such as FRET patent claims of Idaho Technology, Inc., under any patent owned by Roche or AB. under anypatent claim for an apparatus or system, or to use this product for any other purpose or commercial services of any kind.TRADEMARKSThe trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.TaqMan, AmpliTaq Gold, and AmpErase are registered trademarks of Roche Molecular Systems, Inc. TaqMan is used under permission and license. 2011 Life Technologies Corporation. All rights reserved.

ContentsAbout This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Purpose of the guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7User attention words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 CHAPTER 1Product Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Purpose of the Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Description of Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Storage and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Materials Required but Not Supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 CHAPTER 2PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Designing Custom Target Sequences for Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Identifying Target Sequence and Amplicon Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Designing Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Selecting an Amplicon Site for cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1313131314Amplifying Custom Target Sequences for Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Ordering Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Quantitating Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 CHAPTER 3Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Reverse Transcription for All Amplicons Except 18S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Two-Step RT-PCR RT Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Thermal Cycling Parameters for RT Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Performing RT Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .171717181818Reverse Transcription for the 18S Amplicon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Recommended Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Thermal Cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1919191919192021SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide3

Contents CHAPTER 4Optimizing Primer Concentrations . . . . . . . . . . . . . . . . . . 23Optimizing Primer Concentrations for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Optimizing Primer Concentrations for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PCR Master Mix for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Plate Configuration for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Thermal Cycling Parameters for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Confirm the Absence of Nonspecific Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23232324242525Optimizing Primer Concentrations for One-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reducing Nonspecific Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Optimizing Primer Concentrations for One-Step PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .One-Step RT-PCR Master Mix for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Plate Configuration for Primer Optimization for One-Step RT-PCR . . . . . . . . . . . . . . . . . . . . .Thermal Cycling Parameters for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Confirm the Absence of Nonspecific Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2525252626272727Optimizing Primer Concentrations for Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Optimizing Primer Concentrations for Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Two-Step RT-PCR Master Mix for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Plate Configuration for Primer Optimization for Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . .Thermal Cycling Parameters for Primer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Confirm the Absence of Nonspecific Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28282828292929 CHAPTER 5Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Absolute and Relative Quantitation of Target DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Absolute Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Quantitation of cDNA Relative to a Calibrator Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Passive Reference ROX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Terms Used in Quantitation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .313131313132Interpreting the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Adjusting the Baseline and Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 APPENDIX ASupplemental Information . . . . . . . . . . . . . . . . . . . . . . . . 37Preventing Contamination and Nonspecific Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Hot Start PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AmpliTaq Gold DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .False Positives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Optional Use of AmpErase UNG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Fluorescent Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prevention of PCR Product Carryover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General PCR Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4373737373838383839SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide

ContentsAmplicon-Independent Amplification (Including Primer-Dimers) . . . . . . . . . . . . . . . . . . . . . . . . . . .Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Melt Curve Defined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Using Melt Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Using Agarose Gels to Check PCR Product Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . APPENDIX B3939394040Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide5

Contents6SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide

About This GuideIMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.Purpose of the guideThis guide describes how to perform PCR and One-Step or Two-Step RT-PCR usingSYBR Green PCR Master Mix.User attention wordsFive user attention words may appear in this document. Each word implies aparticular level of observation or action as described below:Note: Provides information that may be of interest or help but is not critical to the useof the product.IMPORTANT! Provides information that is necessary for proper instrument operationor accurate chemistry kit use.CAUTION! Indicates a potentially hazardous situation that, if not avoided, mayresult in minor or moderate injury. It may also be used to alert against unsafepractices.WARNING! Indicates a potentially hazardous situation that, if not avoided,could result in death or serious injury.DANGER! Indicates an imminently hazardous situation that, if not avoided,will result in death or serious injury.SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide7

About This GuideUser attention words8SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide

1Product InformationPurpose of the KitThe SYBR Green PCR Master Mix is a convenient premix of the components (exceptprimers, template and water) necessary to perform real-time PCR using SYBR Green IDye. Direct detection of PCR product is monitored by measuring the increase influorescence caused by the binding of SYBR Green dye to double-stranded (ds) DNA.You can perform One-Step or Two-Step RT-PCR using the SYBR Green RT-PCRReagents Kit (see “Materials Required but Not Supplied” on page 10).In RNA quantitation assays, you use the SYBR Green PCR Master Mix in the secondstep of a two-step reverse-transcription polymerase chain reaction (RT-PCR) protocol.In a One-Step RT-PCR protocol, MultiScribe Reverse Transcriptase and RNaseInhibitor are added to the SYBR Green PCR Master Mix.The SYBR Green PCR Master Mix is designed for use with Applied Biosystems realtime PCR systems.For the best quantitation results, use the following: Primer Express software for primer design Applied Biosystems reagents Applied Biosystems universal thermal cycling conditionsMaterials and EquipmentDescription ofMaster MixThe SYBR Green PCR Master Mix is supplied in a 2X concentration and containssufficient reagents to perform 200 50-µL reactions. The mix is optimized for SYBR Green reactions and contains SYBR Green I Dye, AmpliTaq Gold DNA Polymerase,dNTPs with dUTP, Passive Reference, and optimized buffer components.For SYBR Green reagent-based real-time PCR and One-Step or Two-Step RT-PCR, thefollowing components are available:KitP/NContentsSYBR Green PCR MasterMix4309155SYBR Green PCR Master Mix (200 50 μLreactions)SYBR Green RT-PCRReagents Kit4310179SYBR Green PCR Master Mix (200 50 μLreactions)TaqMan Reverse Transcription Reagents(200 10 μL reactions)User Guide4310251—SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide9

1Chapter 1 Product InformationMaterials and EquipmentStorage andStabilityUpon receipt, store the SYBR Green PCR Master Mix at 2 to 8 C and TaqMan Reverse Transcription Reagents at –15 C to –25 C.Note: If stored under the recommended conditions, we guarantee productperformance through the expiration date (control date) printed on the label.Materials Requiredbut Not SuppliedThe items listed in the following tables are required in addition to the reagentssupplied in the SYBR Green PCR Master Mix.For the Safety Data Sheet (SDS) of any chemical not distributed by Life Technologies,contact the chemical manufacturer. Before handling any chemicals, refer to the SDSprovided by the manufacturer, and observe all relevant precautions.ItemAmpErase Uracil-N-glycosylase (UNG)Life Technologies(PN N808-0096)Applied Biosystems Real-Time PCR SystemLife TechnologiesApplied Biosystems Spectral Calibration Kitfor your real-time PCR systemLife TechnologiesMicroAmp 96-well Tray/Retainer Set, 10setsLife Technologies(PN 403081)MicroAmp Cap Installing Tool (Handle)Life Technologies(PN 4330015)MicroAmp Optical 384-Well Reaction Platewith Barcode, 50 platesLife Technologies(PN 4309849)MicroAmp Optical 8-Cap Strip, 300 stripsLife Technologies(PN 4323032)MicroAmp Optical 96-Well Reaction Platewith Barcode and Optical Caps, 20 plateswith capsLife Technologies(PN 403012)MicroAmp Optical Adhesive Film KitLife Technologies(PN 4313663)MicroAmp Optical Tube without Cap,0.2 mL, 2000 tubesLife Technologies(PN N801-0933)Primer Express Software:Life Technologies 5-user license (PN 4363993) 1-user license (PN 4363991)MicroAmp Life Technologies(PN N801-0560)Optical 96-well Reaction PlateNote: The MicroAmp Optical 96-wellReaction Plate may be sealed withMicroAmp Optical Caps or MicroAmp Optical Adhesive Film.10SourceCentrifuge with adapter for 96-well plates orfor 384-well platesMajor laboratory supplier (MLS)Disposable glovesMLSMicrocentrifugeMLSSYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide

Chapter 1 Product InformationMaterials and EquipmentItem1SourceLonza Reliant 4% NuSieve 3:1 Plus AgaroseGel, for DNA 1 kb, supplier number 54928MLSPipette tips, with filter plugsMLSPipettors, positive-displacement or airdisplacementMLSPolypropylene tubesMLSTris-EDTA (TE) Buf

SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide 9 1 Product Information Purpose of the Kit The SYBR Green PCR Master Mix is a convenient premix of the components (except primers, template and water) necessary to perform real-time PCR using SYBR Green I Dye. Direct detection