PCR Essentials - Michigan State Universitycore.phmtox.msu.edu/Scheduling/ItemDocs/13/PCR_Essentials.pdfThe GeneAmp® PCR System 9700 is a high-performance thermal cycler with built-in

PRODUCT BROCHURE PCR Essentials

PCR EssentialsThe world’s most trusted PCR solutions

2

Sequencing and

Resequencing

Cloning Fast PCR Expression Analysis Methylation Multiplex PCR Genotyping Bacterial Gene

Amplification

Viral Gene Amplification Long PCR (5–20 kb)

PCR APPLICATIONS GUIDE

Use direct (noncloning) sequencing for:

•Mutationdetection

•Candidategeneanalysis

•Geneticlinkagestudies

•Evolutionarystudies

•Genome(gapfillingandshotgun) sequencing

•Primerwalking

PCRisthepreferredmethod to obtain a higher abundance of target DNA to clone. Downstream applicationsusinghigh-purityclonedgenesinclude:

•cDNAlibraryconstruction

•Genefamilycharacterization

•Large-scalegenome mappingusingYACs/BACs

PCRcanbeperformedfaster for unique PCR screeningapplications.Suchapplicationsinclude:

•High-throughputscreening

•ColonyPCRlibraryscreening

•Rapidgenotypingoftransgenic animals

UseRT-PCRtoconvertRNAintocDNA,thenamplifythecDNA.Applicationsinclude:

•RapidamplificationofcDNA ends (RACE) to amplifyunknowncDNA

•DifferentialdisplayPCR

•Detectionofraretranscripts

•Quantitativegeneexpression

Studyepigeneticeffectsbyamplificationofbisulfate-treatedDNA:

•SpecificamplificationoflongCpGislands

•Difficulttargets

UsemultiplexPCRtoamplifymanytargetsinasingle tube. This method providestarget-specificamplificationwith primersspecificforeachtarget. Used for:

•Humanidentificationstudies

•Variablenumberoftandemrepeats(VNTR)screening

UsePCRtostudydifferences in DNA sequences. Related applicationsinclude:

•Allele-specificPCR

•Fragment-lengthpolymorphismanalysis

•Haplotyping

•LinkingemulsionPCR

•SNPanalysis

•Microsatellitestudies

UsePCRtoanalyzebacterial genomes— vitalforantibioticandvaccinedevelopment.Alsouseful for:

•Pathogendetection

•Amplifiedfragment-lengthpolymorphism(AFLP)analysisusedincomparativebacterialgenomics

PCRassaysareahighlysensitive,specific,andfast means of detecting virusesfor:

•Nucleicacidsequence-basedamplification(NASBA)


Anenzymeblendefficientlyamplifieslargertargets for:

•Sequencemapping

•MitochondrialgenomePCR

•Geneclusterstudies

•cDNAcloningoflongtranscripts

RECOMMENDED ENZYMES

AmpliTaqGold®FastPCRMasterMix,AmpliTaq® andAmpliTaqGold® 360DNAPolymerases,GeneAmp®High-FidelityPCRSystem

AmpliTaq®andAmpliTaqGold® 360 DNA Polymerase,AmpliTaqGold®360MasterMix,GeneAmp®High-FidelityPCRSystem

AmpliTaqGold® FastPCRMasterMix,GeneAmp® FastPCRMasterMix

GeneAmp®GoldRNAPCRKit,HighCapacityRNA-to-cDNAMasterMix,HighCapacityRNA-to-cDNAKit

AmpliTaqGold® 360 MasterMix,DNAPolymerase,AmpliTaq® 360DNAPolymerase

AmpliTaqGold®FastPCRMasterMix,AmpliTaqGold® 360DNAPolymerase,AmpliTaqGold®360MasterMix

AmpliTaqGold®FastPCRMasterMix,AmpliTaq® 360, AmpliTaqGold® 360

AmpliTaqGold®FastPCRMasterMix,AmpliTaqGold® 360 DNA Polymerase,AmpliTaqGold®360MasterMix

AmpliTaq®andAmpliTaqGold® 360 DNA Polymerases

GeneAmp®High-FidelityPCRSystem,rTthDNAPolymerase,XL

COMPATIBLE THERMAL CYCLERS

Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers


Veriti®96-WellThermalCycler








AppliedBiosystemshelpedtorevolutionizemolecularbiology

withthefirstcommerciallyavailablethermalcyclersandPCR

enzymes.Today,ourcompletelineofPCRproducts—enzymes,

reagents,plastics,andthermalcyclers—areusedinthousandsof

laboratorieseachday.ThankyouformakingourPCRproductsthe

mostwidelyusedintheworld.

3

Sequencing and

Resequencing

Cloning Fast PCR Expression Analysis Methylation Multiplex PCR Genotyping Bacterial Gene

Amplification

Viral Gene Amplification Long PCR (5–20 kb)

PCR APPLICATIONS GUIDE

Use direct (noncloning) sequencing for:

•Mutationdetection

•Candidategeneanalysis

•Geneticlinkagestudies

•Evolutionarystudies

•Genome(gapfillingandshotgun) sequencing

•Primerwalking

PCRisthepreferredmethod to obtain a higher abundance of target DNA to clone. Downstream applicationsusinghigh-purityclonedgenesinclude:

•cDNAlibraryconstruction

•Genefamilycharacterization

•Large-scalegenome mappingusingYACs/BACs

PCRcanbeperformedfaster for unique PCR screeningapplications.Suchapplicationsinclude:

•High-throughputscreening

•ColonyPCRlibraryscreening

•Rapidgenotypingoftransgenic animals

UseRT-PCRtoconvertRNAintocDNA,thenamplifythecDNA.Applicationsinclude:

•RapidamplificationofcDNA ends (RACE) to amplifyunknowncDNA

•DifferentialdisplayPCR

•Detectionofraretranscripts

•Quantitativegeneexpression

Studyepigeneticeffectsbyamplificationofbisulfate-treatedDNA:

•SpecificamplificationoflongCpGislands

•Difficulttargets

UsemultiplexPCRtoamplifymanytargetsinasingle tube. This method providestarget-specificamplificationwith primersspecificforeachtarget. Used for:

•Humanidentificationstudies

•Variablenumberoftandemrepeats(VNTR)screening

UsePCRtostudydifferences in DNA sequences. Related applicationsinclude:

•Allele-specificPCR

•Fragment-lengthpolymorphismanalysis

•Haplotyping

•LinkingemulsionPCR

•SNPanalysis

•Microsatellitestudies

UsePCRtoanalyzebacterial genomes— vitalforantibioticandvaccinedevelopment.Alsouseful for:


•Amplifiedfragment-lengthpolymorphism(AFLP)analysisusedincomparativebacterialgenomics

PCRassaysareahighlysensitive,specific,andfast means of detecting virusesfor:

•Nucleicacidsequence-basedamplification(NASBA)


Anenzymeblendefficientlyamplifieslargertargets for:

•Sequencemapping

•MitochondrialgenomePCR

•Geneclusterstudies

•cDNAcloningoflongtranscripts

RECOMMENDED ENZYMES

AmpliTaqGold®FastPCRMasterMix,AmpliTaq® andAmpliTaqGold® 360DNAPolymerases,GeneAmp®High-FidelityPCRSystem

AmpliTaq®andAmpliTaqGold® 360 DNA Polymerase,AmpliTaqGold®360MasterMix,GeneAmp®High-FidelityPCRSystem

AmpliTaqGold® FastPCRMasterMix,GeneAmp® FastPCRMasterMix

GeneAmp®GoldRNAPCRKit,HighCapacityRNA-to-cDNAMasterMix,HighCapacityRNA-to-cDNAKit

AmpliTaqGold® 360 MasterMix,DNAPolymerase,AmpliTaq® 360DNAPolymerase

AmpliTaqGold®FastPCRMasterMix,AmpliTaqGold® 360DNAPolymerase,AmpliTaqGold®360MasterMix

AmpliTaqGold®FastPCRMasterMix,AmpliTaq® 360, AmpliTaqGold® 360

AmpliTaqGold®FastPCRMasterMix,AmpliTaqGold® 360 DNA Polymerase,AmpliTaqGold®360MasterMix

AmpliTaq®andAmpliTaqGold® 360 DNA Polymerases

GeneAmp®High-FidelityPCRSystem,rTthDNAPolymerase,XL

COMPATIBLE THERMAL CYCLERS



Veriti®96-WellThermalCycler








4

Veriti®

PCR InstrumentsSincetheintroductionofourfirstthermalcyclerin1987, AppliedBiosystemshascontinuedtodevelopandsupport newPCRinstrumentstoempoweryourresearch.

ThePowerofMOREThe Veriti® Thermal Cycler—designed to

meet your current and future PCR needs.

TheVeriti®ThermalCyclerdeliverstheprovenreliabilityyou

expectfromAppliedBiosystemswithenhancedfeaturesto

handleallofyourPCRapplications.Fourformatsareavailable

tomeetyourthermalcyclingneeds:Veriti®96-Well(0.2or0.1

mL),384-Well,or0.5mL60-Well.Anintuitivecolortouchscreen

interfaceprovidesyouwithapowerfulyetsimpleuserinterface

tosimplifyinstrumentsetupanduse.

Additionally,theVeriti®96-WellThermalCyclerprovidesyouwith

theflexibilitytorunFastorstandardPCRaswellasthecapabiltyto

performbetter-than-gradientPCRoptimizationwithVeriFlex™Blocks.

• SixtemperatureblocksforPCRoptimizationonVeriti®96-WellThermalCyclers

• OptimalforfastandstandardPCR

• Reliabilityyouexpect

• Exceptionallyeasytooperate

• OptionalVeritiLink™RemoteManagementSoftwareallowsyoutonetworkandmanageyoursystems,providingyouwithpeaceofmindwithfeatureslikeemailnotifications

EconomicalBenchtopInstrumentThe Applied Biosystems® 2720 Thermal Cycler—

your personal thermal cycler.

Thepersonal-sized2720ThermalCyclerisidealforbasic

PCRandcyclesequencingapplications.Itincorporates

manyfeaturesoftheGeneAmp®PCRSystem9700

toensuresimilarperformanceandreliabilityinamore

compactpackageandatalowerprice.

• Compactdesignmaximizesbenchspace

• Precise and uniform heating and cooling

• Convenientgraphicalinterface

• Reliableandcosteffective

2720

5

9700

PCR INSTRUMENT SELECTION GUIDE

Format Veriti® 96-Well System

Veriti® 384-Well System

Veriti® 60-Well System

96-Well 9700 Dual 96-Well 9700

Dual 384-Well 9700

60-Well 9700 2720

Sample Block 0.1mLor0.2mLAlloyblocks(non-interchangeable)

0.02mLAluminum

0.5mLAluminum

0.2mLAluminum, Silver,orGold-platedsilver

0.2mLAluminum (2x96-Well)

0.02mLAluminum (2x384-Well)

0.5mLAluminum

0.2mLAluminum

Function PCRoptimization,FastPCR,standardPCR

High-throughput384-wellisothermal sampleblock

60-well(0.5mL)isothermal sampleblockforlargerpost-PCRsamplevolumes

Mostflexibleresearch format

Medium-/high-throughputdual96-wellsampleblocksenable192samplesperrun

High-throughput,smallsamplevolume

Largerpost-PCRsamplevolumes

96-wellpersonalizedcycler

Features VeriFlex™Blocks:6peltierblocksforPCRoptimizationortorunuptosix annealing temperaturesinthesame run

VeritiLink™ networkingcapabilityprovidescontrolof12instruments from a single instrument

Supports0.5mLthin-walledtubesfor better heat transfer and moreefficientcycling

Standard0.2mLformat and sampleblockoptionsprovideenhanced performance anddurability

High-throughputdual96-wellsampleblocksenable192samplesperrun

High-throughputdual384-wellsampleblocksenable768samplesperrun,optionalauto-lid

Supports0.5mLthin-walledtubesfor better heat transfer and moreefficientcycling

Exceptionallysmallfootprintdesigned with ventsintherearallowingseveralcyclerstobeplacedside-by-sidetoconservevaluablebenchspace

Temperature Accuracy

±0.25ºC(35ºC–99.9ºC) ±0.5ºC (35ºC–100ºC)

Temperature Range

4ºCto99ºC

Dimensions Height:24.5cm(9.6in) Width:23.7cm(9.3in) Depth:48.5cm(19.1in)

Height: 26cm(10in) Width: 30cm(12in) Depth: 40.6cm(16in)

Height:26cm(10in) Width:30cm(12in) Depth:52cm(20.5in)

Height: 26cm(10in) Width: 30cm(12in) Depth: 40.6cm(16in)

Height: 22cm(8.7in) Width: 21cm(8.3in) Depth: 36cm(14.2in)

Part Numbers

4375305(0.1mL), 4375786(0.2mL)

4388444 4384638 4314879,N8050001,4314878

4343176 N8050002,4314487

4310899 4359659

InterchangeableBlockDesignMeet wide-ranging needs with the flexible

GeneAmp® PCR System 9700.

TheGeneAmp®PCRSystem9700isahigh-performance

thermalcyclerwithbuilt-inflexibilityprovidedbydifferentblock

temperaturemodesandarangeofuser-interchangeablePCR

blockoptions.

• Convenientgraphicalinterface

• Interchangeableblocksavailable:60-well,0.5mL;96-well,0.2mL;anddual96-or384-wellformats

• Optionalrobotics-compatibleautolidavailablewithdual384-wellblock

6

PCREnzymesTrusttheworld’smostreferencedbrandofDNApolymeraseforyourPCRreactions.ReferencestoAmpliTaq®andAmpliTaqGold® DNA Polymerasesappearinmorethan20,000peer-reviewedjournals.AndnowwiththenewAmpliTaq®360productline,thebestjustgotbetter.

PCR ENZYME SELECTION GUIDE

Enzyme Amplify Wide Target Range

Amplicon Size

Specificity Yield Fidelity Convenience Speed

AmpliTaq®360DNAPolymerase •••• ≤5kb •••• •••• ••• ••• ••

AmpliTaqGold®360DNAPolymerase •••• ≤5kb ••••• •••• ••• •••• ••

AmpliTaqGold®360MasterMix ••••• ≤5kb ••••• ••••• ••• ••••• •••

AmpliTaqGold®FastPCRMasterMix,UP •••• ≤2kb ••• •••• ••• ••••• •••••

GeneAmp®FastPCRMasterMix •••• ≤2kb ••• •••• ••• ••••• •••••

GeneAmp®HighFidelityPCRSystem ••• ≤10kb •••• ••••• ••••• ••• •

AmpliTaq®DNAPolymerase,StoffelFragment ••• ≤2kb ••• ••• ••• •• •••

True Allele® PCR Premix ••• ≤5kb •••• ••• ••• •••• •

rTthDNAPolymerase ••• ≤5kb ••• •••• ••• •• ••

rTthDNAPolymerase,XL ••• ≤40kb •••• ••••• ••••• •• ••

PCRforEverydayNeedsAmpliTaq® 360 and AmpliTaq Gold® 360 DNA Polymerases take your routine PCR further.

ThenewAmpliTaq®360andAmpliTaqGold®DNAPolymeraseshavebeenoptimizedtoamplifyawiderangeoftargetswhile

deliveringthereliabilityyouhavecometoexpectfromtraditionalAmpliTaq®DNAPolymerase.

PanelAshowsproductsamplifiedwithAmpliTaqGold®360PCRMasterMixwhilePanelBshowsthesameproductsamplifiedusingtheQIAGENHotStarTaq®MasterMix.PCRreactionswereperformedusing1ngoftemplateDNAperreactionusingcyclingconditionsaccordingtoeachmanufacturer’srecommendations.Allenzymeconcentrationswerestandardizedat0.025U/µL.Annealingtemperatureswereuniformacrosstheselectedtargets.Ampliconsrangedfrom300to1,400basepairs(bp)inlengthwithanaveragelengthof553bp.Eachreactionwasperformedinduplicate.Ampliconsarelabeledasfollows:E=EasyAmplification;A=HighAT;G=HighGC,L=Long;D=PrimerDimer;H=Homopolymer;SQ=SequencingChallenge.ThehighGCamplicon(G)reactionsincluded5µLof360GCEnhancer.

Figure 1. AmpliTaqGold®360MasterMixAmplifiesaBroadRangeofTargets.

• ReliablyamplifyabroadrangeoftargetswhichcontainhighGC,highAT, homopolymerruns,andmore

• Optional360GCSolutionallowsyoutotacklethemostchallengingGC-richfragments with ease

• SuperioryieldallowsyoutodomorewithyourPCRamplicons

• Fewerfalsepositivesandreducedbacterialbackground

• Obtainreproducibleresultsfromthe most trusted source for PCR

• Superiorsequencingresultsevenwithdifficulttargets

A1 A2 A3 A4 A5 A6 E1 E2 E3 E4 D1

D2 D3 D4 D5 D6 H1 H2 H3 H4 H5 H6

H15 SQ1 SQ2 SQ3 SQ4 SQ5 SQ6 SQ7 SQ8 SQ9 L1

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11

A1 A2 A3 A4 A5 A6 E1 E2 E3 E4 D1

D2 D3 D4 D5 D6 H1 H2 H3 H4 H5 H6


G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11

Panel A. AmpliTaq Gold® 360 Master Mix

Panel B. QIAGEN HotStarTaq® Master Mix

A1 A2 A3 A4 A5 A6 E1 E2 E3 E4 D1

D2 D3 D4 D5 D6 H1 H2 H3 H4 H5 H6


G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11

A1 A2 A3 A4 A5 A6 E1 E2 E3 E4 D1

D2 D3 D4 D5 D6 H1 H2 H3 H4 H5 H6


G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11

Panel A. AmpliTaq Gold® 360 Master Mix

Panel B. QIAGEN HotStarTaq® Master Mix

PanelA.AmpliTaqGold®360MasterMix PanelB.QIAGENHotStarTaq®MasterMix

7

Hot-StartPCRforHigh SpecificityandYieldAmplify the broadest range of targets with

AmpliTaq Gold® 360 Master Mix and DNA Polymerase.

TheAmpliTaqGold®360MasterMixhasbeenoptimizedto

amplifyawiderangeoftargetswithoutcompromisingon

specificity,yield,sensitivity,orreproducibility.

• ReliablyamplifythebroadestrangeoftargetswhichcontainhighGC,highAT,homopolymerruns,andmore

• IncreaseyourconfidenceinobtainingPCRresultsthefirsttimeandeverytime

• 360GCEnhancerallowsyoutotacklethemostchallengingGC-richfragmentswithease

•MasterMixprovidessuperioryieldandconveniencesoyoucandomorewithyourPCRamplicons

• Fewerfalsepositivesandreducedbacterialbackground

• Obtainreproducibleresultsusingourhighlystringenthot-startenzymewithaproventrackrecord

• Superiorsequencingresultsevenwithdifficulttargets

• Availableasaconvenientmastermixorinkitformat

FastPCRwithHot-StartSpecificityUse AmpliTaq Gold® Fast PCR Master Mix, UP

for fast results in your sequencing applications.

AmpliTaqGold®FastPCRMasterMix,UPreducesyourtime

toresultswhiledeliveringspecific,high-yieldampliconsfor

easyproductionofhigh-qualitysequencingdata.

• Usesyourexistingprimerstoamplifystandard600bptargetsfromgenomic DNA in about 40 minutes

• WhencombinedwithmodifiedcyclesequencingconditionsforFastcyclesequencing,youcango fromsampletobasecallinginapproximately4hours

• Sensitiveenoughtodetectupto10copiesofasinglegenein10ngofgenomicDNA

Use GeneAmp® Fast PCR Master Mix for Fast

PCR on sequences up to 2 kb in <50 minutes.

Decreaseyourturnaroundtimebyreducingamplificationtimes

byapproximately1/3overconventionalPCRreactiontimes.

• Amplifiesstandard500bptargetsfromgenomicDNAbackgroundinunder25minutes

• Detectssingle-copygenesin10ngofhumangenomicDNA

Combine these convenient master mixes with the Applied Biosystems® Veriti® 96-Well Fast Thermal Cycler(page 4) for faster PCR than ever before.

LongTemplatesandIncreasedFidelityforAccuratePCRAchieve high fidelity and yield with the

GeneAmp® High-Fidelity PCR System.

TheGeneAmp®High-FidelityPCRSystemisdesignedfor

accurate,high-yieldPCR.Itcontainsanenzymeblendof

AmpliTaq®DNAPolymeraseandathermostableproofreading

enzyme.ThisproprietaryblendincreasesthefidelityofPCR

threefoldoverTaq DNApolymerasealone.

• Enzymeblendisformulatedforincreasedfidelityandyield

• ProofreadingactivityenablesamplificationofDNAfragmentsupto10kb

• Two-buffersystemprovidesversatility

For longer targets–up to 40 kb–use rTth DNA Polymerase, XL.

Figure 2. RobustResultswithAppliedBiosystems®PCRMasterMixes.

VarioustargettypesincludinghighAT,GCrich,primerdimer,homopolyermer,short,andlongwereamplifiedontheVeriti®96-WellFastThermalCycler,usingathree-stepprotocol.Eachampliconwasamplifiedfrom10ngofhumangenomicDNA.Amplificationtimesfora600basepairfragmentvariedasfollows:40minforAmpliTaqGold® FastPCRMasterMix,UP,25minforGeneAmp®FastPCRMasterMix,and2hoursforthestandardAmpliTaqGold®PCRMasterMix.

High AT

GC Rich

Primer D

imer

Homopolymer

EasyLong

Ladder (bp)

3% Gel, 150V, 20 min

800400

200

100

AmpliTaqGold®FastPCRMasterMix,UP

GeneAmp® FastPCR

MasterMix

AmpliTaqGold® PCR MasterMix

8

ReverseTranscriptase(RT) PCREnzymes

5 10 15 20 25 30 35 40 45

45

40

35

30

25

20

15

10

5

High Capacity RNA-to-cDNA Master Mix CT

Qua

ntiT

ect®

Rev

erse

Tran

scrip

tion

Kit

(Qia

gen)

Figure 3. ComparisonofHighCapacityRNA-to-cDNAMasterMixwithaCompetitorProduct.TheHighCapacityRNA-to-cDNAMasterMixwascomparedwithQuantiTectReverseTranscriptionKit(Qiagen),acrossapanelof96TaqMan®GeneExpressionAssays.TheCTvaluesofthecompetitorproductisdeemedequivalentifthedatamarkerislocatedonthe45°line.Datamarkerslocatedabovethe45°linearearesultofthecompetitorproductyieldingadelayedCTvalueorpoorerresultscomparedtotheHighCapacityRNA-to-cDNAMasterMix.RNAwastranscribedin20µLreactions,accordingtothemanufacturers’recommendedprotocols.ThecDNAreactionswerepooledandaliquotsusedinfourreal-timePCRreactionswiththeTaqMan®GeneExpressionMasterMixandoneofthe96TaqMan®assays.

Figure 4. Amplificationofß-actinfromTotalHumanRNA.

Lanes DescriptionLaneM: Molecularweightmarker Lanes3-6: GeneAmp®GoldRNAPCRKit,includingMultiScribe™ andAmpliTaqGold®DNAPolymerasewithautomated hot start Lanes7-10: RNAPCRwithMuLVandTaq DNAPolymerase, no hot start Lanes11-14: RNAPCRwithAMVandenzymeblend,nohotstart

TotalRNA(pg) 1001020100102010010 2 0

FirstStrandcDNASynthesisfor GeneExpressionStreamline cDNA synthesis with High Capacity

RNA-to-cDNA Master Mix for high-throughput or

High Capacity RNA-to-cDNA Kit for everyday use.

ForfastandefficientqualitativeconversionoftotalRNAtocDNAinasinglereaction,weofferyoutwocompletecDNAsynthesiskitstosuityourthroughputneeds.Eachprovidesexceptionallinearityatlowconcentrationsandstreamlinesyourworkflow,speedingyourtimetoresults.

High Capacity RNA-to-cDNA Master Mix

•5Xconcentrationprovidedinasingletubeorpre-dispensedin96-wellplateformat,alsoavailablewithoptional “No-RT”Controlsforassayvalidation

•DeliversrobustresultsevenwithdiluteRNA

High Capacity RNA-to-cDNA Kit

•Streamlinedworkflowwithtwo-tubeformatforeverydayuse

•Reliablereversetranscriptionofabundantandlimitedtargets

Whetheryou’reseekingtheflexibilityoftwo-stepRT-PCR orthespeedofone-stepRT-PCR,ourreversetranscriptases providetheefficiencyandreliabilityyouneed.

Hot-StartRT-PCRfor HighSpecificityandYieldPerform one- or two-step RT-PCR with the

GeneAmp® Gold RNA PCR Reagent Kit.

TheoptimizedGeneAmp®GoldRNAPCRReagent

KitcombinestheflexibilityofMultiScribe™Reverse

TranscriptasewiththespecificityofAmpliTaqGold® DNA

Polymerase.Thesetwoenzymescomplementeachother

toproducedramaticresults.

•Multiplexingoptiontosavetimeandmaterials

• Flexiblechemistryforone-ortwo-stepreactions

• Chemicallymodifiedhot-startDNApolymeraseforhigherspecificity

• Time-releaseDNApolymeraseforgreatersensitivitywithdifficulttemplates

9

Additional PCR Reagents

NucleotidesGeneAmp® dNTPs

GeneAmp®dNTPsaredeoxynucleotidetriphosphates

dissolvedinglass-distilledwaterandtitratedtopH7.0

withNaOH.Availablepackagedindividuallyorblendedin

a dNTP mix.

CarryoverPreventionReagentsAmpErase® UNG

Eliminatefalsepositivescausedbyproductscarried

overfrompreviousPCRrunsbyusingAmpErase®

UNGandGeneAmp®dNTPswithdUTP.AmpErase®

UNGhelpsdegradedU-containingPCRproducts

frompreviousPCRreactions,whileleavingstandard

dT-containingDNAuntouched.Usefulforclinicallabs

andanylabamplifyingthesametargetfrommultiple

DNAsamples.

GeneAmp® dNTPs with dUTP

UsedUTPtogeneratedU-containingPCRproductsin

acarryoverpreventionstrategyalongwithAmpErase®

uracilN-glycosylase(UNG).

AspartofourcompletePCR solution,AppliedBiosystemsoffers ultra-purenucleotidestopreventcarryovercontamination.

Figure 5. RNAPCRProductsUsingtheGeneAmp® EZ rTth RNA PCR Kit. AllreactionswereperformedusingthesameRT-PCRconditionsandPCRtemperatureprofiles.Reactionswereincubatedfor30minutesat65°Cforreversetranscription,followedbytwo-temperaturePCRamplificationfor40cycles.ElectrophoreticanalysisofPCRproducts(10%oftotalreactionvolume)wasperformedona1%agarosegelstained with ethidium bromide.

Target length: 171217225 246258420 308308244142142bp

(bp)516394298200

142

M M-CSF

PDGF-B

PDGF-A

HMG

LDL-R

IL-1 + - HCV

HIV - M

SensitiveRNADetectionfor QuantitationandScreeningDetect low-level cellular RNA and viruses with the

GeneAmp® EZ rTth RNA PCR Kit.

IntheGeneAmp® EZ rTth RNA PCR Kit, rTth DNA

Polymeraseservesasbothreversetranscriptaseand

DNApolymerase.Theuniquesingle-buffersystemis

specificallydesignedforrapidRNAPCRscreening,and

isideallysuitedfordetectionandquantitationofcellular

andviralRNA.

•Minimizescarryovercontaminationusingarobustone-step,one-enzymeprotocol

• HightemperatureRTisidealforRNAtargetswithhighGCcontentorsecondarystructure

• Generatesavisiblebandfrom0.25fg(500copies)pAW109RNA

10

Plastic ConsumablesMicroAmp®disposablesareoptimizedtoprovideunmatchedtemperatureaccuracyanduniformityforfast,efficientPCRamplification.TheplasticsarecompatiblewithvariousAppliedBiosystems®platforms,includingPCRinstruments.Chooseyourformatbasedonthroughputrequirementsor yourthermalcyclerblock.

96-WellReactionPlateswithBarcodeProcess multiple samples in parallel with

MicroAmp® Optical 96-Well Reaction Plates with Barcode.

MicroAmp®reactionplatesminimize

light interference and distribute heat

uniformlyandquickly.

• Frostedplasticminimizesinterferingfluorescencefromcyclingblock

• Optimizedforwell-to-welltemperatureuniformity

• ValidatedonAppliedBiosystems®real-timePCRsystems

384-WellReactionPlateswithBarcodeOptimized for use with Applied Biosystems® end-point and real-time PCR systems.

Theseplatescomplementreal-timePCR

systemsandthedual384-WellGeneAmp®

PCRSystem9700.Thebarcodeshelp

eliminatetrackingerrors,andtheoptical

propertiesoftheplatesreplicatethoseof

otherMicroAmp®opticalplastics.

• Bestplatetouseforreal-timePCR

• Bestplateforhigh-throughputPCR

ReactionTubesforSmall-ScaleStudiesEasily identify individual samples using colored MicroAmp® Reaction Tubes with caps.

MicroAmp®ReactionTubesuniformly

distributeheatforprecisePCRresults.

Availableincolorless,red,orange,blue,and

green, these tubes stand out in a crowd.

• Captivelidwithpositive-clickclosureassuresproperseating

• Polished surface and conical bottomensuremaximumsamplerecovery

• Optionofautoclavabletubesallowsclean, controlled start

• Availableineconomicalbulkpackages

11

PLASTIC CONSUMABLES COMPATIBILITY CHART FOR APPLIED BIOSYSTEMS® INSTRUMENTS

End-point PCR Systems Real-Time PCR Systems Genetic Analyzers

Category/Part Description P/N Veriti® 96-Well 0.1 mL

Veriti® 96-Well 0.2 mL

Veriti® 384-Well

60-Well Veriti®/ 9700 (60-well)

27xx 6100 9700 (96-well)

9700 (384-well)

StepOne™ StepOnePlus™ 7000 7300/ 7500

7500 Fast

7900HT (96-well)

7900HT (384-well)

7900HT Fast

310* 3100/3130

3700/3730

96-well Plates

MicroAmp® Fast 96-Well Reaction Plate (0.1 mL) -10 plates 4346907 x x x

MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL) - 20 plates

4346906 x x x x x x

MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL) - 200 plates

4366932 x x x x x x

MicroAmp® Optical 96-Well Reaction Plate - 10 plates N8010560 x x x x x x x x

MicroAmp® Optical 96-Well Reaction Plate - 500 plates 4316813 x x x x x x x x

MicroAmp® Optical 96-Well Reaction Plate with Barcode - 20 plates 4306737 x x x x x x x x

MicroAmp® Optical 96-Well Reaction Plate with Barcode - 500 plates 4326659 x x x x x x x x

MicroAmp® Optical 96-Well Reaction Plate with Barcode and Optical Caps - 20 plates

403012 x x x x x x

MicroAmp® Optical 96-Well Reaction Plate with Barcode and Optical Adhesive Films - 100 plates

4314320 x x x x x x

384-well Plates

MicroAmp® Optical 384-Well Reaction Plate with Barcode - 50 plates 4309849 x x x x

MicroAmp® Optical 384-Well Reaction Plate with Barcode - 500 plates 4326270 x x x x

MicroAmp® Optical 384-Well Reaction Plate with Barcode- 1,000 plates 4343814 x x x x

MicroAmp® Optical 384-Well Reaction Plate - 1,000 plates 4343370 x x x x

48-well Plates

MicroAmp® Fast Optical 48-Well Reaction Plate - 20 plates 4375816 x x

Single Tubes

MicroAmp® Fast Reaction Tube with Cap (0.1mL) - 1,000 tubes 4358297 x x x

MicroAmp® Reaction Tube with Cap (0.2 mL) - 1,000 tubes N8010540 x x x

MicroAmp® Reaction Tube with Cap (0.2 mL) - 10,000 tubes N8011540 x x x

MicroAmp® Reaction Tube with Cap, Assorted Colors (0.2 mL) - 1,000 tubes N8010840 x x x

MicroAmp® Reaction Tube with Cap, Autoclaved (0.2 mL) - 1,000 tubes N8010612 x x x

MicroAmp® Reaction Tube without Cap (0.2 mL) - 2,000 tubes N8010533 x x x

MicroAmp® Reaction Tube without Cap (0.2 mL) - 10,000 tubes N8011533 x x x

MicroAmp® Reaction Tube without Cap, Assorted Colors (0.2 mL) - 1,000 tubes N8010833 x x x

MicroAmp® Optical Tube without Cap (0.2mL) - 2,000 tubes N8010933 x x x x x

GeneAmp® Thin-Walled Reaction Tube with Flat Cap (0.5 mL) - 1,000 tubes N8010737 x

GeneAmp® Thin-Walled Reaction Tube with Domed Cap (0.5 mL) - 2,000 tubes N8010537 x

GeneAmp® Thin-Walled Reaction Tube with Domed Cap, Autoclaved (0.5 mL) -1,000 tubes

N8010611 x

MicroAmp® 96-Well Reaction Tube/Tray/Retainer Set (0.2 mL) - 20 trays 403083 x x

MicroAmp® 96-Well Reaction Tube/Tray/Retainer Set (0.2 mL) - 100 trays 403086 x x

Strip Tubes and Caps

MicroAmp® 12-Cap Strip - 200 strips N8010534 x x x x

MicroAmp® 12-Cap Strip - 1,000 strips N8011534 x x x x

MicroAmp® 12-Cap Strip, Assorted Colors - 200 strips N8010834 x x x x x

MicroAmp® Fast 8-Tube Strip (0.1 mL) - 125 strips 4358293 x x x

MicroAmp® 8-Tube Strip (0.2 mL)- 125 strips N8010580 x x x x

MicroAmp® 8-Tube Strip, Assorted Colors (0.2 mL) - 120 strips N8010838 x x x x x

MicroAmp® Optical 8-Tube Strip (0.2 mL) -1,000 strips 4316567 x x x x x x

MicroAmp® 8-Cap Strip - 300 strips N8010535 x x x x

MicroAmp® 8-Cap Strip- 1,500 strips N8011535 x x x x

MicroAmp® 8-Cap Strip, Assorted Colors - 300 strips N8010835 x x x x

MicroAmp® Optical 8-Cap Strip - 300 strips 4323032 x x x x x x x x x

Seals & Covers

MicroAmp® Clear Adhesive Film - 100 films 4306311 x x x x x

MicroAmp® Optical Adhesive Film - 25 films 4360954 x x x x x x x x x x x x

MicroAmp® Optical Adhesive Film - 100 films 4311971 x x x x x x x x x x x x

MicroAmp® 48-Well Optical Adhesive Film - 100 films 4375323 x x

MicroAmp® 48-Well Optical Adhesive Film - 25 films 4375928 x x

MicroAmp® Optical Adhesive Film Kit - 20 kits 4313663 x x x x

MicroAmp® 96-Well Full Plate Cover - 5 covers N8010550 x

Reaction Trays

MicroAmp® 96-Well Tray/Retainer Set - 10 trays 403081 x x x

MicroAmp® 96-Well Tray - 10 trays N8010541 x x

MicroAmp® 96-Well Tray for VeriFlex™ Blocks - 10 trays 4379983 x x x

MicroAmp® 96-Well Tray/Retainer Set for Veriti® Systems - 10 trays 4381850 x

MicroAmp® Fast 48-Well Tray - 10 trays 4375282 x

Compression Pads

MicroAmp® Optical Film Compression Pad - 5 pads 4312639 x x

MicroAmp® Snap-On Optical Film Compression Pad - 9 pads 4333292 x

*Requirestheuseofproperadapters.

12

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

NOTICETOPURCHASER:PLEASEREFERTOTHEUSER’SGUIDEORPACKAGEINSERTOFTHEPRODUCTSNAMEDHEREINFORLIMITEDLABELLICENSEORDISCLAIMERINFORMATION.

©2009LifeTechnologiesCorporation.Allrightsreserved.ThetrademarksmentionedhereinarethepropertyofLifeTechnologiesCorporationortheirrespectiveowners.AmpErase,AmpliTaq,andAmpliTaqGoldareregisteredtrademarksofRocheMolecularSystems,Inc.Informationsubjecttochangewithoutnotice.

PrintedintheUSA06/2009Publication104BR04-05

ORDER INFORMATION

Description Quantity Part #

Thermal Cyclers AppliedBiosystems®Veriti®96-WellFastThermalCycler,0.1mL 1instrument 4375305

AppliedBiosystems®Veriti®96-WellThermalCycler,0.2mL 1instrument 4375786



VeritiLink™RemoteManagementSoftware 1disc 4393565

Gold96-WellGeneAmp®PCRSystem9700 Basemodule+sampleblock 4314878

Silver96-WellGeneAmp®PCRSystem9700 Basemodule+sampleblock N8050001

Aluminum96-WellGeneAmp®PCRSystem9700 Basemodule+sampleblock 4314879

Dual96-WellGeneAmp®PCRSystem9700 Basemodule+sampleblock 4343176

Dual384-WellGeneAmp®PCRSystem9700 Basemodule+sampleblock N8050002

Auto-LidDual384-WellGeneAmp®PCRSystem9700 Basemodule+sampleblock 4314487

AppliedBiosystems®2720ThermalCycler 1instrument 4359659

PCR Enzymes, Nucleotides, and Reagents AmpliTaqGold®360MasterMix 1mL/5mL 4398787/4398790

AmpliTaqGold®360DNAPolymerase 100units/250units 4398757/4398760

AmpliTaq®360DNAPolymerase 100units/250units 4398745/4398748

AmpliTaqGold®FastPCRMasterMix,UP 250reactions/2500reactions 4390939/4390941

GeneAmp®FastPCRMasterMix(2X)withProtocol 2x1.25mL 4362070

GeneAmp®High-FidelityPCRSystem 40 reactions 4328216

rTthDNAPolymerase,XL 400 units N8080187

GeneAmp®dNTPBlend,10mM 1mL N8080260

AmpErase®UNG 100units N8080096

RT-PCR Enzymes and Reagents GeneAmp®GoldRNAPCRReagentKit 1kit 4308206

HighCapacityRNA-to-cDNAMasterMixwith“No-RT”Controls 50reactions/200reactions 4390777/4390778

HighCapacityRNA-to-cDNAKit 50reactions 4387406

Plastics for Veriti® 96-Well (0.2 ml), 9700 and 2720 Thermal Cyclers MicroAmp®Optical96-WellReactionPlatewithBarcode 20plates 4306737

MicroAmp®8-TubeStrip(0.2mL) 125strips N8010580

MicroAmp®ReactionTubewithCap(0.2mL) 1,000tubes N8010540

MicroAmp®ClearAdhesiveFilm 100films 4306311

MicroAmp®12-CapStrip 200strips N8010534


MicroAmp®96-WellTrayforVeriFlex™Blocks* 10trays 4379983

MicroAmp®96-WellTray/RetainerSetforVeriti®Systems* 10trays 4381850

MicroAmp®96-WellTray/RetainerSet 10trays 403081

Plastics for Veriti® 96-Well (0.1 ml) Thermal Cycler MicroAmp®FastOptical96-WellReactionPlatewithBarcode(0.1mL) 20plates 4346906

MicroAmp®Fast8-TubeStrip(0.1mL) 125strips 4358293

MicroAmp®FastReactionTubewithCap(0.1mL) 1,000tubes 4358297

MicroAmp®ClearAdhesiveFilm 100films 4306311



MicroAmp®96-WellTrayforVeriFlex™Blocks* 10trays 4379983

Plastics for Veriti® and 9700 384-Well Thermal Cyclers and Veriti® and 9700 60-Well Thermal Cyclers MicroAmp®Optical384-WellReactionPlatewithBarcode 50plates 4309849

GeneAmp®Thin-WalledReactionTubewithFlatCap(0.5mL) 1,000tubes N8010737

GeneAmp®Thin-WalledReactionTubewithDomedCap(0.5mL) 2,000 tubes N8010537

*For Veriti® 96-Well Thermal Cycler VeriFlex™ Blocks only

For complete lists visit www.appliedbiosystems.com/pcr.

Documents

PCR Essentials - Michigan State Universitycore.phmtox.msu.edu/Scheduling/ItemDocs/13/PCR_Essentials.pdfThe GeneAmp® PCR System 9700 is a high-performance thermal cycler with built-in