Master GREEN 200 Manual de usuario Pagina 1

SYBR® Green PCR Master Mix and RT-PCRProtocol

1-4 IntroductionABI PRISM® 7000 Sequence Detection Systems Spectral Calibration KitApplied Biosystems(P/N 4328895)ABI PRISM™ Cap Installing Tool Appli

Introduction 1-5Storage andStabilityUpon receipt, store the SYBR Green PCR Master Mix at 2 to 8 °C and TaqMan Reverse Transcription Reagents at –20 °C

1-6 IntroductionSafetyDocumentationUser AttentionWordsFive user attention words appear in the text of all Applied Biosystems user documentation. Each

Introduction 1-7Chemical WasteHazard WarningCHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can

1-8 IntroductionOrdering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the

Introduction 1-9Preventing Contamination and Nonspecific AmplificationOverviewThe DNA amplification capability of the PCR process makes special labora

1-10 IntroductionFalse PositivesSpecial laboratory practices are necessary in order to avoid false positive amplifications (Higuchi, et al., 1989). Th

Introduction 1-11be purchased individually (P/N N808-0096) or as part of the SYBR® Green Core Reagents Kit (P/N 4304886).FluorescentContaminantsSince

1-12 IntroductionChange gloves whenever you suspect that they are contaminated.Maintain separate areas and dedicated equipment and supplies for: – Sam

Introduction 1-13Amplicon Independent Amplification (Including Primer-Dimer)IntroductionThis section discusses the use of dissociation curves to detec

© Copyright 2002, Applied Biosystems All rights reserved.For Research Use Only. Not for use in diagnostic procedures.NOTICE TO PURCHASER: LIMITED LICE

1-14 IntroductionWhen to Generate Dissociation CurvesThe GeneAmp® 5700 SDS, ABI PRISM® 7900HT SDS, ABI PRISM® 7700 SDS, and ABI PRISM® 7000 SDS can be

PCR 2-1PCR 2OverviewAbout ThisChapterThis chapter describes how to design and amplify custom target sequences for quantitation.In This ChapterThe foll

2-2 PCRDesigning Custom Target Sequences for QuantitationOverviewWe recommend the following steps to design custom primers and identify target sequenc

PCR 2-3.Selecting anAmplicon Site forGenomic DNAOverviewSelecting a good amplicon site ensures amplification of the target mRNA without co-amplifying

2-4 PCRAmplifying Custom Target Sequences for QuantitationOverviewWe recommend the following steps for the development of real-time quantitative PCR a

Reverse Transcription 3-1Reverse Transcription 3OverviewAbout ThisChapterThis chapter provides procedures for performing reverse transcription (RT).In

3-2 Reverse TranscriptionReverse Transcription for All Amplicons Except 18SOverviewSynthesis of cDNA from total RNA samples is the first step in the t

Reverse Transcription 3-3Two-Step RT-PCRRT Reaction MixRT Reaction Mix ComponentVolume/Tube (µL) Final ConcentrationRNase-free water See belowaa. The

3-4 Reverse TranscriptionPerforming RTReactionsThe procedure for generating cDNA using the TaqMan Reverse Transcription Reagents is described below.CH

Reverse Transcription 3-5Reverse Transcription for the 18S AmpliconOverviewSynthesis of cDNA from total RNA samples is the first step in the two-step

Contentsiii1 IntroductionOverview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1About Thi

3-6 Reverse TranscriptionGuidelinesFollow the guidelines below to ensure optimal RT performance.Poly A+ RNA samples are not recommended for 18S experi

Reverse Transcription 3-7CHEMICAL HAZARD. TaqMan Reverse Transcription Reagents may cause eye and skin irritation. They may cause discomfort if swallo

3-8 Reverse Transcription6 Centrifuge the tubes briefly to eliminate air bubbles in the mixture. 7 Label four 0.2-mL MicroAmp Reaction Tubes for the f

Reverse Transcription 3-9Thermal CyclingTo conduct RT thermal cycling:Step Action1 Load the reactions into a thermal cycler.2 Program your thermal cyc

Optimizing Primer Concentrations 4-1Optimizing Primer Concentrations 4OverviewAbout ThisChapterThis chapter describes how to optimize primer concentra

4-2 Optimizing Primer ConcentrationsOptimizing Primer Concentrations for PCR OverviewThe purpose of the procedure below is to determine the minimum pr

Optimizing Primer Concentrations 4-3PCR Master Mixfor PrimerOptimizationCHEMICAL HAZARD. SYBR Green may cause eye, skin, and respiratory tract irritat

4-4 Optimizing Primer ConcentrationsThermal CyclingParameters forPrimerOptimizationIMPORTANT The 10 min, 95 °C step is required to activate the AmpliT

Optimizing Primer Concentrations 4-5Optimizing Primer Concentrations for One-Step RT-PCROverviewThe procedure below is used to optimize one-step RT-PC

ivAmplicon Independent Amplification (Including Primer-Dimer) . . . . . . . 1-13Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4-6 Optimizing Primer ConcentrationsOne-Step RT-PCRMaster Mix forPrimerOptimizationCHEMICAL HAZARD. SYBR Green may cause eye, skin, and respiratory tr

Optimizing Primer Concentrations 4-7Plate Configuration for Primer Optimization for One-Step RT-PCR WellsPCR Master Mix + RT Reagents (µL)a5 µM Forwar

4-8 Optimizing Primer ConcentrationsConfirm theAbsence ofNonspecificAmplificationThermal Cycling Parameters for Primer Optimization StepRTAmpliTaq Gol

Optimizing Primer Concentrations 4-9Optimizing Primer Concentrations for Two-Step RT-PCROverviewThe purpose of the procedure below is to determine the

4-10 Optimizing Primer ConcentrationsTwo-Step RT-PCRMaster Mix forPrimerOptimizationCHEMICAL HAZARD. SYBR Green may cause eye, skin, and respiratory t

Optimizing Primer Concentrations 4-11Confirm theAbsence ofNonspecificAmplificationE5–E8 25 3.0 3.0 0 19.0 50E9–E12 25 3.0 9.0 0 13.0 50F1–F4 25 9.0 0.

Data Analysis 5-1Data Analysis 5OverviewAbout ThisChapterThe chapter describes how to analyze the data generated in your experiment.In This ChapterThe

5-2 Data AnalysisAbsolute and Relative Quantitation of Target DNAOverviewTwo types of quantitation are possible when using the SYBR Green® PCR Master

Data Analysis 5-3Interpreting the ResultsPassive ReferenceROXThe Passive Reference (ROX) is a dye molecule included in the SYBR Green PCR Master Mix t

vTemplate Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5Guidelines . . . . . . . . . . . . . . . . . . .

References A-1References AFaloona, F., Weiss, S., Ferre, F., and Mullis, K. 1990. Direct detection of HIV sequences in blood high-gain polymerase chai

Technical Support B-1Technical Support BServices & SupportAppliedBiosystems WebSiteTo access the Applied Biosystems Web site, go to:http://www.app

Headquarters850 Lincoln Centre DriveFoster City, CA 94404 USAPhone: +1 650.638.5800Toll Free (In North America): +1 800.345.5224Fax: +1 650.638.5884Wo

viAbsolute Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2Quantitation of cDNA Relative to a Calibrator Sam

Introduction 1-1Introduction 1OverviewAbout ThisChapterThis chapter describes the SYBR® Green PCR Master Mix and provides important safety information

1-2 IntroductionPurpose of the KitAbout the KitThe SYBR Green PCR Master Mix is a convenient premix of all the components, except primers, template an

Introduction 1-3Materials and EquipmentDescription ofMaster MixThe SYBR Green PCR Master Mix is supplied in a 2X concentration and contains sufficient