Post on 11-Jul-2020
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)
•Pathogendetection
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®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®96-WellThermalCycler
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
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:
•Pathogendetection
•Amplifiedfragment-lengthpolymorphism(AFLP)analysisusedincomparativebacterialgenomics
PCRassaysareahighlysensitive,specific,andfast means of detecting virusesfor:
•Nucleicacidsequence-basedamplification(NASBA)
•Pathogendetection
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®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®96-WellThermalCycler
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
Veriti®ThermalCyclers,GeneAmp®PCRSystem9700, and 2720 Thermal Cyclers
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
H15 SQ1 SQ2 SQ3 SQ4 SQ5 SQ6 SQ7 SQ8 SQ9 L1
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
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
H15 SQ1 SQ2 SQ3 SQ4 SQ5 SQ6 SQ7 SQ8 SQ9 L1
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
Headquarters850LincolnCentreDrive |FosterCity,CA94404USAPhone650.638.5800|TollFree800.345.5224www.appliedbiosystems.com
International SalesForourofficelocationspleasecallthedivision headquartersorrefertoourWebsiteat www.appliedbiosystems.com/about/offices.cfm
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
AppliedBiosystems®Veriti®384-WellThermalCycler,0.02mL 1instrument 4388444
AppliedBiosystems®Veriti®60-WellThermalCycler,0.5mL 1instrument 4384638
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®8-CapStrip 300strips N8010535
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®12-CapStrip 200strips N8010534
MicroAmp®8-CapStrip 300strips N8010535
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.