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Table of contents
I. Description..................................................................................................................2
II. Principle........................................................................................................................2
III. Content.........................................................................................................................3
IV. Storage..........................................................................................................................4
V. Features........................................................................................................................4
VI. PrecautionsforUse..................................................................................................4
VII. Protocol........................................................................................................................4
A)ProtocolusingSmartCycler®IISystem...................................................4
B)ProtocolusingABIPRISM®7000/7700/7900HT&
7300/7500/7500FastReal-TimePCRSystem.......................................7
C)ProtocolusingLightCycler®.........................................................................9
D)ProtocolusingMx3000P............................................................................ 12
VIII. ApplicationExample............................................................................................ 14
IX. Relatedproducts.................................................................................................... 14
1
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
I. DescriptionPremixExTaq™(PerfectRealTime)isa2×convenientpremixreagent,speciallydesignedforrealtimePCRbyusingTaqMan®* 1probe.ThisproductcombinesthehighperformanceofTaKaRaExTaq™HS,whichisanenzymeforhotstartPCRutilizingTaqantibody,withanewlydevelopedbufferwhichprovidessuperiorspecificity,increasedamplificationefficiencyandhighaptitudeforhigh-speedrealtimePCR.Accordingly,asuccessfulrealtimePCRisprom-isedwithhighsensitivity,widedynamicrangeandaccuratequantification.
ApplicablerealtimePCRinstruments;・ ABIPRISM®7000,7700,7900HT,*2 AppliedBiosystems7300,7500Real-TimePCRSystem, 7500FastReal-TimePCRSystem(LifeTechnologies)・ LightCycler®(RocheDiagnostics)*3・ SmartCycler®System/SmartCycler®IISystem(Cepheid)*4・ Mx3000P(Stratagene)・ ThermalCyslerDice™RealTimeSystemII (TaKaRaBio)*5
* 1:TaqMan®isatrademarkofRocheMolecularSystemsInc.* 2:ABIPRISM®isaregisteredtrademarkofAppleraCorporation.* 3:LightCycler®isaregisteredtrademarkofRocheDiegnostics.* 4:SmartCycler®isatrademarkofCepheid.* 5:ThisinstrumentisnotavailableintheU.S.andEurope.
II. PrincipleThisproductemploysTaKaRaExTaq™HSforPCRreaction.PCRproductsaredetectedwithTaqMan®probeinrealtimemonitoring.
1)PCRPCR(PolymeraseChainReaction)isasimpleandpowerfulmethodtoamplifyonlyatargetDNAofatinyamountbycyclingthreeincubationstepsatdifferenttemperatures:Double-strandedtargetDNAisheatdenatured(denaturationstep),thetwoprimerscomplementarytothetargetsegmentareannealedatlowtemperature(annealingstep),andtheannealedprimersarethenextendedatanintermediatetemperature(extensionstep)withaDNApolymerase.Asthetargetcopynumberdoublesuponeachcycle,PCRcantherebyamplifyDNAfragmentsupto106-foldinashortperiod.AsthisproductutilizesanenzymeforHotStartPCR,TaKaRaExTaq™HS,non-specificam-plificationduetomisprimingpriortocyclingorduetoprimerdimmercanbeminimized.Accordinglythehighlyspecificandsensitivedetectionisachieved.
2)Fluorescencedetection:TaqMan®Probemethod:TheTaqMan®methodisbasedonacombinationofTaqMan®TechnologyandarealtimePCRinstrument.Oligonucleotidemodifiedwithfluorescencesubstance(e.g.FAM)at5'-endandwithquencher(e.g.TAMRA)at3'-endisaddedinareactionsystem.Undertheannealingcondition,TaqMan®probespecificallyhybridizestotemplateDNA,buttheflu-orescenceissuppressedbyquencher.Inextensionstep,the5'→3'exonucleaseactivityofTaqDNApolymerasedegradestheTaqMan®probehybridizedtoatemplate.Accordingly,TaqMan®probeisreleasedfromthesuppressionwithaquencher,resultinginemissionoffluorescence.Bymeasuringthefluorescenceintensitytheamountofamplifiedproductcanbemonitored.
2
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
III. Content (250 units)PremixExTaq™(PerfectRealTime)(2×conc.)*1 1.0ml×5tubesROXReferenceDye(50×conc.)*2 200μlROXReferenceDyeII(50×conc.)*2 200μl
* 1:containsTaKaRaExTaq™HS,dNTPMixture,andMg2+.* 2:ROX™ReferenceDye/DyeIIisusedfornormalizationofintensitybybackgroundsubtrac-ROX™ReferenceDye/DyeIIisusedfornormalizationofintensitybybackgroundsubtrac-
tion.ForABIPRISM®7000/7700/7900HTandAppliedBiosystems7300Real-TimePCRSystem,theuseofROX™ReferenceDye(50×)isrecommended.ForAppliedBiosystems7500Real-TimePCRSystem,7500FastReal-TimePCRSystem,andStratageneMx3000P,theuseofROX™ReferenceDyeIIisrecommended.
ItisnotrequiredforusewithSmartCycler®,LightCycler®orThermalCyclerDice™RealTimeSystemII .
Reagents and Instruments Required but Not Supplied in this product
1.ThermalCyclerforrealtimePCR(authorizedinstruments)2.ReactiontubeorplateforrealtimePCR3.PCRprimers4.TaqMan®probefordetection(licensedprobes)5.dH2O6.MicropippetsandMicropippettips(autoclavedpriortouse)
QF
Q
Q
F
F
F Q
2)Primerannealing/Probehybridization
Polymerase
Primer
ProbeFluorescenceSubstance
Quencher
Hybridizes
1)Heatdenaturation
3)Extension
3
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
IV. StorageStableat4℃for6months.Carefulattentionshouldbemadenottocausecontamination.
*Thisproductisshippedat-20℃.Afterdelivered,storeat4℃.Maketheconcentra-Thisproductisshippedat-20℃.Afterdelivered,storeat4℃.Maketheconcentra--20℃.Afterdelivered,storeat4℃.Maketheconcentra-20℃.Afterdelivered,storeat4℃.Maketheconcentra-tionuniformbygentlyinvertingatubebeforeuse.
*Thisproductcanbestoredat-20℃forlongtermstorage.Oncethawed,storeat4℃anduseupwithin6months.
V. Features1) Quick&accuratedetectionandquantificationoftargetgenethroughrealtimePCR.2) Easy-to-use2×premixreagentdesignedforreducingpipettingsteps.3) Highamplificationefficiencyandhighlysensitivedetection:Thisproductutilizesanenzymeforhotstart,TaKaRaExTaq™HS.Asthisenzyme-buffersystemisoptimizedforrealtimePCR,thisproductoffershighlyefficientamplificationandhighsensitivedetection.
VI. Precautions for UsePleaseensuretoreadthroughthefollowingprecautionspriortostartingtheprotocol.
1) Priortouse,maketheconcentrationuniformbygentlyinvertingatubebeforeuse.2) Aprecipiatemayforminthisproductduringstorageat-20℃.Thisprecipitatecanbecompletelydissolvedbybrieflywarmingthetubewithyourhandsorplacingthetubeatroomtemperature,followedbyinversionofthetubeseveraltimestomixuntildissolved.USETHEREAGENTONLYAFTERCOMPLETELYREDISSOLVINGTHEPRECIPITATEtoensureauniformconcentrationofcomponents.Donotvortex.Evenintheabsenceofaprecipitate,gentlemixingoftheproduct(avoidingairbubbles)isrecommendedtoprovideauniformconcentrationofcomponentspriortouse.
3) TaqMan®probefordetectionshouldbepreparedseparately.Itisnotsuppliedinthisproduct.
4) Forpreparinganddispensingthereagents,anewdisposabletipshouldbeusedtominimizecontaminationamongsamples.
VII. ProtocolA) Protocol using Smart Cycler® II System1.PreparethefollowingPCRreactionmixture.<perreaction>[Reagents] [Volume] [Finalconcentration]PremixExTaq™(2×) 12.5μl 1×PCRForwardPrimer(10μM) 0.5μl 0.2μM*1PCRReversePrimer(10μM) 0.5μl 0.2μM*1TaqMan®probe 1μl *2
template 2μl *3
dH2O 8.5μltotal 25μl
4
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
* 1:Thefinalconcentrationofprimerscanbe0.2μMinmostreactions.Whenitdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
* 2:ProbeconcentrationdiffersdependingonkindofrealtimePCRinstrument,andkindoffluorescencelabelingmaterial.Theaddingamountshouldbede-terminedbyreferringtotheoperationmanualofinstrumentandaproductinsertsuppliedwithprobe.GenerallywhenperformingdetectionwithSmartCycler®System,probeconcentrationshouldbedeterminedtohavethefinalconcentrationwithintherange0.1-0.5μM.
* 3:Finaltemplateconcentrationvariesdependingonthecopynumberoftargetpresentinthetemplatesolution.Optimalamountshouldbede-terminedbypreparingthedilutionseries.DNAtemplateshouldbeusedinlessthan100ng.WhentheRTreactant(cDNA)isusedasatemplate,itshouldbeaddedinless10%volumeofPCRreactionmixture.
2.Startthereaction.GentlycentrifugethereactiontubesusingthecentrifugeexclusiveuseforSmartCycler®.LoadthereactiontubesonSmartCycler®IISystemandstartthereaction.ShuttlePCRstandardprotocol(below)isrecommended.Trythisprotocolfirstly,andoptimizethereactionconditionifneeded.
ShuttlePCRStandardProtocolStage1:InitialDenaturation Stage2:PCRreaction
Note : ThisproductcombinesthehighperformanceofTaKaRaExTaq™HS,whichisanenzymeforhotstartPCRutilizingTaqantibody.InitialdenaturationsteppriortoPCRshouldbeat95℃for30sec.Noneedtoheatat95℃for(5-)15min.astheinitialdenaturation,requiredforchemicallymodifiedTaqpolymerase.Ifexcessiveheattreatmentisprovided,theenzymeactivitydescreasesandtheamplificationef-ficiencyandtheaccuracyinquantificationcanalsobeaffected.
5
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
InitialDenaturationStep Temperature Time Detection Remark
InitialDenaturation 95℃ 30sec. OFF
Itisrecommendedtoperforminitialdenaturationat95℃for30seconds,whichisenoughevenwhenthetemplateisgenomicDNAorcircularplasmid.Dena-turationconditionthatexceeds2minutesunstabilizesthereactionthusnotrecommended.
ShuttlePCRStep Temperature Time Detection Remark
Denaturation 95℃ 3-5sec. OFF
GeneralamplifiedsizeforrealtimePCRislessthan300bp,sodena-turationat95℃for3-5secondswouldbesufficient.
Annealing/Extension 56-64℃ 20-30sec. ON
Pleasetryat60℃for20secondsatfirst.Thetemperatureshouldbeoptimizedwithintherangeof56-64℃ifoptimizationisrequired.Whenreactiondoesnotproceedefficiently,extendthetime.
Cyclenumber;30-45cycles
3.Afterthereactioniscompleted,performanalysis.Afterthereactioniscompleted,verifytheamplificationcurve.Establishthestandardcurvewhenquantificationisdone.
* RefertotheoperationmanualofSmartCycler®fortheanalysismethodwithSmartCycler®.
6
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
B) Protocol using ABI PRISM 7000/7700/7900HT or Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
1.PreparePCRreaction(below).
<perreaction>[Reagents] [Volume] [Volume] [Finalconc.]PremixExTaq™(2×) 10μl 25μl 1×PCRForwardPrimer(10μM) 0.4μl 1μl 0.2μM* 1PCRReversePrimer(10μM) 0.4μl 1μl 0.2μM* 1TaqMan®probe 0.8μl 2μl * 2
ROXReferenceDyeorDyeII(50×) * 3 0.4μl 1μl 1×template 2μl 4μl * 4
dH2O 6μl 16μltotal 20μl * 5 50μl * 5
* 1:Thefinalconcentrationofprimerscanbe0.2μMinmostreactions.Whenitdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
* 2:ProbeconcentrationdiffersdependingonkindofrealtimePCRinstrument,andkindoffluorescencelabelingmaterial.Theaddingamountshouldbedeter-minedbyreferringtotheoperationmanualofinstrumentandaproductinsertsuppliedwithprobe.
* 3:TheROX™ReferenceDye/DyeIIissuppliedforperformingnormalizationoffluo-3:TheROX™ReferenceDye/DyeIIissuppliedforperformingnormalizationoffluo-rescentsignalintensitiesamongwellswhenusedwithrealtimePCRinstrumentsthathaveoption.ForABIPRISM®7000/7700/7900HTandAppliedBiosystems7300Real-TimePCRSyatem,theuseofROX™ReferenceDye(50×)isrecom-mended.ForAppliedBiosystems7500Real-TimePCRSystemand7500FastReal-TimePCRSystem,theuseofROX™ReferenceDyeIIisrecommended.
* 4:Finaltemplateconcentrationvariesdependingonthecopynumberoftargetpresentinthetemplatesolution.Optimalamountshouldbedeterminedbypreparingthedilutionseries.DNAtemplateshouldbeusedinlessthan100ngfor20μlreaction.WhentheRTreactant(cDNA)isusedasatemplate,itshouldbeaddedinless10%volumeofPCRreactionmixture.
* 5:Thereactionof50μlisfor96-wellplate,singletubeand8-striptube.Thereactionof20μlisfor384-wellplateand96-wellFastThemalCyclingPlate.
2.Startthereaction.ShuttlePCRstandardprotocol(below)isrecommended.Trythisprotocolfirstly,andoptimizethereactionconditionifneeded.
Note : ThisproductcombinesthehighperformanceofTaKaRaExTaq™HS,whichisanenzymeforhotstartPCRutilizingTaqantibody.Initialdena-turationsteppriortoPCRshouldbeat95℃for30sec.Noneedtoheatat95℃for(5-)15min.astheinitialdenaturation,requiredforchemi--)15min.astheinitialdenaturation,requiredforchemi-)15min.astheinitialdenaturation,requiredforchemi-callymodifiedTaqpolymerase.Ifexcessiveheattreatmentisprovided,theenzymeactivitydescreasesandtheamplificationefficiencyandtheaccuracyinquantificationcanalsobeaffected.
7
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
ShuttlePCRStandardProtocol
Stage1:InitialdenaturationReps:195℃30sec.
Stage2:PCRReps:4095℃5sec.60℃30sec.(31sec,34sec) *
*: Fluorescencedetectionstepshouldbe30secondswithABIPRISM7700and7900HT,31secondswithABIPRISM7000and7300Real-TimePCRSystem,and34secondswith7500Real-TimePCRSystem.
ShuttlePCRStandardProtocol
HoldingStageReps:195℃,30min.
CyclingStageNumberofCycles:4095℃,3min.60℃,30min.
MeltCurveStage
[7500FastReal-TimePCRSystem]
[ABIPRISM7000/7700/7900HT,7300/7500Real-TimePCRSystem]
8
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
InitialdenaturationstepStep Temperature Time Detection Remark
Initialdenaturation 95℃ 30sec. OFF
Itisrecommendedtoperforminitialdenaturationat95℃for30seconds,whichisenoughevenwhenthetemplateisgenomicDNAorcircularplasmid.Dena-turationconditionthatexceeds2minutesunstabilizesthereactionthusnotrecommended.
ShuttlePCRStep Temperature Time Detection Remark
Denaturation 95℃ 3-5sec. OFF
SincethetargetsizeamplifiedforrealtimePCRisgenerallyshorterthan300bp,thedenaturationat95℃for3-5secondsisasuf--5secondsisasuf-5secondsisasuf-ficientcondition.
Annealing/Extension 56-64℃
25-34sec.(25, 30, 31,34sec.) *
ON
Pleasetryat60℃for30,31,34or25secondsatfirst.Thetemperatureshouldbeoptimizedwithintherangeof56-64℃ifoptimizationisrequired.Whenreactiondoesnotproceedefficiently,extendthetime.
*: Fluorescencedetectionstepshouldbe30secondswithABIPRISM7700and7900HT,31secondswith7000and7300,and34secondswith7500Real-TimePCRSystem.Whenusedwith7500Fast,set25sec.
Cyclenumber;30-45cycles
3. Afterthereactioniscompleted,verifytheamplificationcurve.Establishthestandardcurvewhenquantificationisdone.* RefertotheoperationmanualofanusedrealtimePCRinstrument.
C) Protocol using LightCycler®
1.PreparethefollowingPCRreactionmixture.<perreaction>[Reagents] [Volume] [Finalconcentration]PremixExTaq™(2×) 10μl 1×PCRForwardPrimer(10μM) 0.4μl 0.2μM*1PCRReversePrimer(10μM) 0.4μl 0.2μM*1TaqMan®probe 0.8μl *2
template 2μl *3
dH2O 6.4μltotal 20μl
9
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
* 1:Thefinalconcentrationofprimerscanbe0.2μMinmostreactions.Whenitdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
* 2:ProbeconcentrationdiffersdependingonkindofrealtimePCRinstrument,andkindoffluorescencelabelingmaterial.Theaddingamountshouldbedeter-minedbyreferringtotheoperationmanualofinstrumentandaproductinsertsuppliedwithprobe.
* 3:Finaltemplateconcentrationvariesdependingonthecopynumberoftargetpresentinthetemplatesolution.Optimalamountshouldbedeterminedbypreparingthedilutionseries.DNAtemplateshouldbeusedinlessthan100ng.WhentheRTreactant(cDNA)isusedasatemplate,itshouldbeaddedinless10%volumeofPCRreactionmixture.
2.Startthereaction.AftergentlycentrifugationofLightCycler®capillaries,setcapillariesinaLightCyclerandstartthereaction.ShuttlePCRstandardprotocol(below)isrecommended.Trythisprotocolfirstly,andoptimizethereactionconditionifneeded.
Note : ThisproductcombinesthehighperformanceofTaKaRaExTaq™HS,whichisanenzymeforhotstartPCRutilizingTaqantibody.Initialdena-turationsteppriortoPCRshouldbeat95℃for30sec.Noneedtoheatat95℃for(5-)15min.astheinitialdenaturation,requiredforchemi--)15min.astheinitialdenaturation,requiredforchemi-)15min.astheinitialdenaturation,requiredforchemi-callymodifiedTaqpolymerase.Ifexcessiveheattreatmentisprovided,theenzymeactivitydescreasesandtheamplificationefficiencyandtheaccuracyinquantificationcanalsobeaffected.
ShuttlePCRStandardProtocol
Stage1:InitialdenaturationReps:195℃,30sec.20℃/sec.
Stage2:PCRReps:4095℃,5sec.20℃/sec.60℃,20sec.20℃/sec.
Stage2:PCRreaction
10
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
Initialdenaturation
Step Temperature Time AcquisitionMode Remark
Initialdenaturation 95℃ 30sec. None
Itisrecommendedtoperforminitialdenaturationat95℃for30seconds,whichisenoughevenwhenthetemplateisgenomicDNAorcircularplasmid.Dena-turationconditionthatexceeds2minutesunstabilizesthereactionthusnotrecommended.
ShuttlePCR
Step Temperature Time AcquisitionMode Remark
Denaturation 95℃ 3-5sec. None
SincethetargetsizeamplifiedforrealtimePCRisgenerallyshorterthan300bp,thedenaturationat95℃for3-5secondsisasuf--5secondsisasuf-5secondsisasuf-ficientcondition.
Annealing/Extension 56-64℃ 20-30sec. Single
Pleasetryat60℃for20secondsatfirst.Thetemperatureshouldbeoptimizedwithintherangeof56-64℃ifoptimizationisrequired.Whenreactiondoesnotproceedefficiently,extendthetime.
Cyclenumber:30-45cycles
3. Afterthereactioniscompleted,verifytheamplificationcurve.Establishthestandardcurvewhenquantificationisdone.* RefertotheoperationmanualofanusedrealtimePCRinstrument.
11
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
D) Protocol using Mx3000P
1.PreparethefollowingPCRreactionmixtureonice.
<perreaction>[Reagents] [Volume] [Finalconc.]PremixExTaq™(2×) 12.5μl 1×PCRForwardPrimer(10μM) 0.5μl 0.2μM*1PCRReversePrimer(10μM) 0.5μl 0.2μM*1TaqMan®probe 1μl *2
ROXReferenceDyeII(50×)*3 0.5μl 1×template(<100ng) 2μl *4
dH2O 8.0μltotal 25μl
* 1:Thefinalconcentrationofprimerscanbe0.2μMinmostreactions.Whenitdoesnotwork,determinetheoptimalconcentrationswithintherangeof0.1-1.0μM.
* 2:ProbeconcentrationdiffersdependingonkindofrealtimePCRinstrumentandtypeoffluorescencelabelingmaterial.Theaddingamountshouldbedeter-minedbyreferringtotheoperationmanualofinstrumentandaproductinsertsuppliedwithprobe.
* 3:ForMx3000P,theuseofROXReferenceDyeIIisrecomended.ROXReferenceDyeisnotsuitablefortheusewithMx3000P,sinceithashigherconcentrationthanROXReferenceDyeII.
* 4:Finaltemplateconcentrationvariesdependingonthecopynumberoftargetpresentinthetemplatesolution.Optimalamountshouldbedeterminedbypre-paringthedilutionseries.ItisrecomendedtoapplyDNAtemplateinlessthan100ng.WhentheRTreactant(cDNA)isusedasatemplate,itshouldbeaddedinlessthan10%volumeofPCRreactionmixture.
2.Startthereaction.ShuttlePCRstandardprotocol(below)isrecommended.Trythisprotocolfirstly,andoptimizethereactionconditionifneeded.
ShuttlePCRStandardProtocolStage1:Initialdenaturation Reps:1 95℃,30sec.
Stage2:PCR Reps:40 95℃,5sec. 60℃,20sec.
Note : ThisproductcombinesthehighperformanceofTaKaRaExTaq™HS,whichisanenzymeforhotstartPCRutilizingTaqantibody.Initialdena-turationsteppriortoPCRshouldbeat95℃for30sec.Noneedtoheatat95℃for(5-)15min.astheinitialdenaturation,requiredforchemi--)15min.astheinitialdenaturation,requiredforchemi-)15min.astheinitialdenaturation,requiredforchemi-callymodifiedTaqpolymerase.Ifexcessiveheattreatmentisprovided,theenzymeactivitydescreasesandtheamplificationefficiencyandtheaccuracyinquantificationcanalsobeaffected.
12
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
InitialdenaturationStep Temperature Time Detection Remark
Initialdenaturation 95℃ 30sec. OFF
Itisrecommendedtoperforminitialdenaturationat95℃for30seconds,whichisenoughevenwhenthetemplateisgenomicDNAorcircularplasmid.Dena-turationconditionthatexceeds2minutesunstabilizesthereactionthusnotrecommended.
ShuttlePCRStep Temperature Time Detection Remark
Denaturation 95℃ 3-5sec. OFF
SincethetargetsizeamplifiedforrealtimePCRisgenerallyshorterthan300bp,thedenaturationat95℃for3-5secondsisasuf--5secondsisasuf-5secondsisasuf-ficientcondition.
Annealing/Extension 56-64℃ 20-30sec. ON
Pleasetryat60℃for20secondsatfirst.Thetemperatureshouldbeoptimizedwithintherangeof56-64℃ifoptimizationisrequired.Whenreactiondoesnotproceedefficiently,extendthetime.
Cyclenumber:30-45cycles
3.Afterthereactioniscompleted,performanalysis.Afterthereactioniscompleted,verifytheamplificationcurve.Establishthestandardcurvewhenquatitationisdone.* RefertotheoperationmanualofanusedrealtimePCRinstrument.
13
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
95℃ 30sec.↓95℃ 3sec. 45cycle60℃ 25sec.
Amplificationcurve
R2=0.9998Slope=-3.417
Standardcurve
VIII. Application exampleDetection example with Applied Biosystems 7500 Fast Real-Time PCR System
Target:MouseGapdPrimer/Probe:TaqMan®GeneExpressionAssays(LifeTechnologies)Template:cDNAserialdilution(correspondingtototalRNA500fg-50ng)
IX. Related productsPrimeScript™RTreagentKit(PerfectRealTime)(Cat.#RR037A)OneStepPrimeScript™RT-PCRKit(PerfectRealTime)(Cat.#RR064A)SYBR®PremixExTaq ™II(TliRNaseHPlus)(Cat.#RR820A/B)SYBR®PremixExTaq ™(TliRNaseHPlus)(Cat.#RR420A/B)OneStepSYBR®PrimeScript™RT-PCRKitII(PerfectRealTime)(Cat.#RR086A)OneStepSYBR®PrimeScript™RT-PCRKit(PerfectRealTime)(Cat.#RR066A)
14
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
15
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
NOTICETOPURCHASER:LIMITEDLICENSE
[P7] PCR NoticeAlicensetoperformthepatented5'NucleaseProcessforresearchisobtainedbythepurchaseof(i)bothAuthorized5'NucleaseCoreKitandLicensedProbe,(ii)aLicensed5'NucleaseKit,or(iii)licenserightsfromAppliedBiosystems.
ThisproductisanAuthorized5'NucleaseCoreKit.UseofthisproductiscoveredbyoneormoreofthefollowingUSpatentsandcorrespondingpatentclaimsoutsidetheUS:5,079,352,5,789,224,5,618,711,6,127,155,5,677,152,5,773,258,5,407,800,5,322,770,5,310,652,5,210,015,5,487,972,andclaimsoutsidetheUScorrespondingtoUSPatentNo.4,889,818.Thepurchaseofthisprod-uctincludesalimited,non-transferableimmunityfromsuitundertheforegoingpatentclaimsforusingonlythisamountofproductforthepurchaser'sowninternalresearch.SeparatepurchaseofaLicensedProbewouldconveyrightsundertheapplicableclaimsofUSPatentsNos.5,538,848,5,723,591,5,876,930,6,030,787,6,258,569,5,804,375(claims1-12only),and6,214,979,andcor-respondingclaimsoutsidetheUnitedStates.Norightunderanyotherpatentclaimandnorighttoperformcommercialservicesofanykind,includingwithoutlimitationreportingtheresultsofpurchaser'sactivitiesforafeeorothercommercialconsideration,isconveyedexpressly,byimplication,orbyestoppel.Thisproductisforresearchuseonly.DiagnosticusesunderRochepatentsrequireaseparatelicensefromRoche.FurtherinformationonpurchasinglicensesmaybeobtainedfromtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.
[L15] Hot Start PCRLicensedunderU.S.PatentNo.5.338,671and5,587,287,andcorrespondingpatentsinothercountries.
[L52] Rox Reference Dye (Research Field)Purchaseofthisproductincludesanimmunityfromsuitunderpatentsspecifiedintheproductinserttouseonlytheamountpurchasedforthepurchaser'sowninternalresearch.Nootherpatentrightsareconveyedexpressly,byimplication,orbyestoppel.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.
[M57] LA TechnologyThisproductiscoveredbytheclaims6-16ofU.S.PatentNo.5,436,149anditsforeigncounterpartpatentclaims.
TaqMan®The5'nucleaseprocessiscoveredbypatentsownedbyRocheMolecularSystems,Inc.andF.Hoffmann-LaRocheLtd.Purchaseoftheproductdoesnotprovidealicensetousethispatentedtechnology.
16
Premix Ex Taq™ (Perfect Real Time) Cat.#RR039Av1103Da
URL:http://www.takara-bio.com
NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTAKARABIOINC.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775437247orfromourwebsiteatwww.takara-bio.com.