Table of Contents
I. Description........................................................................................................ 3
II. Principle............................................................................................................. 3
III. Components.................................................................................................... 4
IV. Storage............................................................................................................... 5
V. Features.............................................................................................................. 5
VI. PrecautionsforUse........................................................................................ 5
VII. Protocol.............................................................................................................. 6
VIII. RelatedProducts..........................................................................................14
2
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
I. DescriptionPremixExTaq(PerfectRealTime)isdesignedforprobe-basedqPCR.ThisproductisalsosuitableforfastPCR.PremixExTaqallowsaccuratetargetquantificationanddetectionoverabroaddynamicrangeandmakesitpossibletoobtainhighlyreproducibleandreliablereal-timePCRresults.Theproductissuppliedasa2Xpremixtofacilitateeasypreparationofreactionmixtures.ThecombinationofTaKaRaExTaq®HS,ahotstartPCRenzymethatusesananti-Taqantibody,andabufferoptimizedforreal-timePCRsuppressesnon-specificamplificationandallowshighamplificationefficiencyandhighdetectionsensitivityinreal-timePCRanalyses.
Compatible real-time PCR instruments:・ AppliedBiosystems7300/7500/7500FastReal-TimePCRSystem(ThermoFisherScientific)• LightCycler(RocheDiagnostics)• SmartCyclerSystem/SmartCyclerIISystem(Cepheid)• Mx3000P(AgilentTechnologies)• ThermalCyclerDice™RealTimeSystemIII(Cat.#TP950/TP970/TP980/TP990)*
* Notavailableinallgeographiclocations.Checkforavailabilityinyourarea.
II. PrincipleThisproductemploysTaKaRaExTaqHSforPCRAmplification.PCRAmplificationproductscanbemonitoredinrealtimeusingaprobe.
1) PCRPCR(PolymeraseChainReaction)isasimpleandpowerfulmethodtoamplifyasmallamountoftargetDNAbycyclingatthreedifferenttemperatures;double-strandedtargetDNAisheatdenatured(denaturationstep),twoprimerscomplementarytothetargetsegmentareannealedatlowtemperature(annealingstep),andtheannealedprimersarethenextendedatanintermediatetemperature(extensionstep)withthermostableDNApolymerase.Asthetargetcopynumberdoubleseachcycle,DNAfragmentscanbeamplifiedupto106-foldinashortperiod.
2) Fluorescence detectionOligonucleotidesmodifiedwithfluorescencesubstance(e.g.FAM)atthe5'-endandwithquencher(e.g.TAMRA)atthe3'-endareaddedtothereaction.DuringtheannealingphaseofPCR,theprobespecificallyhybridizeswiththetemplateDNA;however,fluorescenceofthefluorophoreisinhibitedbythepresenceofthenearbyquencher.DuringtheextensionphaseofthePCR,the5'-3'exonucleaseactivityofTaqDNApolymerasedegradestheprobethatishybridizedtothetemplate.Thisresultsinareleaseofquenchersuppressionandfluorescenceemission.Thefluorescenceintensitycorrelateswiththeamountofamplifiedproduct.
3
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
III. Components (200 reactions, 50 μl PCR)PremixExTaq(PerfectRealTime)(2Xconc.)*1 1.0ml x5ROXReferenceDye(50Xconc.)*2 200μlROXReferenceDyeII(50Xconc.)*2 200μl
*1 ContainsTaKaRaExTaqHS,dNTPMixture,andMg2+.*2 Usewhenperforminganalyseswithreal-timePCRinstrumentsthatnormalize
fluorescentsignalsbetweenwells,suchasAppliedBiosystemsinstruments.
◆ AddROXReferenceDye(50X)inavolumeequivalentto1/50ofthePCRreactionmixture:・AppliedBiosystems7300Real-TimePCRSystem(ThermoFisherScientific)
◆ AddROXReferenceDyeII(50X)inavolumeequivalentto1/50ofthePCRreactionmixture:・AppliedBiosystems7500/7500FastReal-TimePCRSystem(ThermoFisherScientific)・Mx3000P(AgilentTechnologies)
◆ NoROXReferenceDyeisrequiredwhenusinganyofthefollowingsystems:・LightCycler(RocheDiagnostics)・SmartCyclerSystem/SmartCyclerIISystem(Cepheid)・ThermalCyclerDiceRealTimeSystemIII(Cat.#TP950/TP970/TP980/TP990)*3
*3 Notavailableinallgeographiclocations.Checkforavailabilityinyourarea.
Materials Required but not Provided-DNAamplificationsystemforreal-timePCR(authorizedinstruments)-ReactiontubesorplatesdesignedspecificallyfortheqPCRinstrumentused-PCRprimers-Probefordetection(TaKaRaqPCRProbe,etc.)-Sterilepurifiedwater-Micropipetteandtips(sterile,withfilter)
3) Extension
Hybridization
PrimerQuencherFluorophore
Probe
Polymerase
1) Heat denaturation
2) Primer annealing/probe hybridization
QF
Q
Q
F
F
F Q
4
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
IV. StorageStoreat4℃(stableforupto6months).
*Protectfromlight;becarefulnottointroducecontamination.
1. Storetheproductat4℃afterreceipt.Beforeuse,gentlyinverttubetomakesurereagentiscompletelydissolvedandevenlymixed.
2. Thisproductmaybefrozenat-20℃forlong-termstorage.Oncethawed,itshouldbestoredat4℃andusedwithin6months.
V. Features1. Quickandaccuratedetectionandquantificationoftargetgeneusingreal-timePCR.2. Easy-to-use2Xpremixreducespipettingsteps.3. Highamplificationefficiencyandhighdetectionsensitivity.
VI. Precautions for UseRead through the following precautions prior to starting the protocol.1. Priortouse,makesurethereagentisthoroughlymixedbygentlyinvertingthetubeseveraltimeswithoutgeneratingbubbles;bubblesmayimpairreactivity.Donotvortex.
Whenstoredat-20℃,PremixExTaq(2X)mayprecipitate.Todissolvetheprecipitantcompletely,placeatroomtemperature(below30℃)briefly,theninvertthetubeseveraltimes.Thepresenceofprecipitateisindicativeofpoorlymixedreagent.Makesurereagentisevenlymixedbeforeuse.
2. Placethereagentoniceimmediatelyafterithasthawed.
3. Thisproductisnotsuppliedwithprimersandprobes.
4. Usenewdisposabletipstominimizepotentialcross-contaminationbetweensampleswhenpreparingreactionmixturesordispensingaliquots.
5. TaKaRaExTaqHSinthispremixisahotstartPCRenzymethatcontainsananti-Taqantibodythatinhibitspolymeraseactivity.Donotperformthepre-PCRincubation(5-15minat95℃)thatisrequiredforothercompanies’chemicallymodifiedhotstartPCRenzymes.Prolongeddenaturationmayinactivatetheenzyme,affectingamplificationefficiencyandquantificationaccuracy.
Fortheinitialdenaturationstep,incubationat95℃for30secisgenerallysufficient.
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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
VII. Protocol1. Protocol for Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
1. PreparePCRreactionmixture.
<Perreaction>Reagent Volume Volume Finalconc.PremixExTaq(2X) 10μl 25μl 1XPCRForwardPrimer(10μM) 0.4μl 1μl 0.2μM*1PCRReversePrimer(10μM) 0.4μl 1μl 0.2μM*1Probe*2 0.8μl 2μlROXReferenceDyeorDyeII(50X)*3 0.4μl 1μl 1Xtemplate*4 2μl 4μlSterilepurifiedwater 6μl 16μlTotal 20μl*5 50μl *5
*1 Thefinalconcentrationofprimersshouldbe0.2μMformostreactions.Ifthisdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
*2 Theprobeconcentrationwilldifferdependingonthespecificreal-timePCRinstrumentandthetypeoffluorescentlabel.Theamountnecessaryshouldbedeterminedbyreferringtotheoperationmanualofinstrumentandtheproductinsertsuppliedwithprobe.
*3 TheROXReferenceDye/DyeIIisintendedfornormalizationoffluorescentsignalintensitiesamongwellswhenusedwithreal-timePCRinstrumentsthathavethisoption.For7300Real-TimePCRSystem,ROXReferenceDye(50X)isrecommended.For7500Real-TimePCRSystemand7500FastReal-TimePCRSystem,ROXReferenceDyeIIisrecommended.
*4 ThefinaltemplateconcentrationvariesdependingonthecopynumberoftargetDNApresentinthetemplatesolution.Theoptimalamountshouldbedeterminedbypreparingadilutionseries.DNAtemplatesshouldbeusedinamountslessthan100ngper20μlreaction.WhenthecDNA(reversetranscriptionsolution)isusedasatemplate,itshouldbeaddedinavolumethatislessthan10%ofthetotalvolumeofthePCRreactionmixture.
*5 Adjustaccordingtotherecommendedvolumeforeachapparatus.
2. Startthereaction. TheshuttlePCRstandardprotocolisrecommended.Trythisprotocolfirst,thenoptimizethereactionconditionsifneeded.(Referto“GeneralOverviewofPCRConditions”onpage13.)
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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
ShuttlePCRStandardProtocolStage1:InitialdenaturationReps:195℃ 30sec
Stage2:PCRReps:4095℃ 5sec60℃ 31or34sec*
* 31secwith7300Real-TimePCRSystem,and34secwith7500Real-TimePCRSystem.
<AppliedBiosystems7300/7500Real-TimePCRSystem>
<AppliedBiosystems7500FastReal-TimePCRSystem>ShuttlePCRStandardProtocolHoldingStageNumberofCycle:195℃ 30sec
CyclingStageNumberofCycles:4095℃ 3sec60℃ 30sec
Note: ThisproductcombinesthehighperformanceofTaKaRaExTaqHS,whichisanenzymeforhotstartPCRthatusesaTaqantibody.InitialdenaturationpriortoPCRshouldbeat95℃for30sec.Thereisnoneedforincubationat95℃for(5-)15minastheinitialdenaturation,suchasrequiredforchemicallymodifiedTaqpolymerases.Excessiveheattreatmentcandecreaseenzymeactivity,andtheamplificationefficiencyandaccuracyinquantificationcanalsobeaffected.
3. Afterreactioncompletion,verifytheamplificationcurves.Establishthestandardcurvewhenabsolutequantificationisperformed.
Refertotheinstructionmanualforthereal-timePCRinstrumentusedforspecificanalysismethods.
7
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
2. Protocol for LightCycler
1. PreparethefollowingPCRreactionmixture.
<Perreaction>Reagent Volume Finalconc.PremixExTaq(2X) 10μl 1XPCRForwardPrimer(10μM) 0.4μl 0.2μM*1PCRReversePrimer(10μM) 0.4μl 0.2μM*1Probe*2 0.8μltemplate(<100ng)*3 2μlSterilepurifiedwater 6.4μlTotal 20μl
*1 Thefinalconcentrationofprimersshouldbe0.2μMformostreactions.Ifthisdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
*2 Theprobeconcentrationwilldifferdependingonthespecificreal-timePCRinstrumentandthetypeoffluorescentlabel.Theamountnecessaryshouldbedeterminedbyreferringtotheoperationmanualofinstrumentandtheproductinsertsuppliedwithprobe.
*3 ThefinaltemplateconcentrationvariesdependingonthecopynumberoftargetDNApresentinthetemplatesolution.Theoptimalamountshouldbedeterminedbypreparingadilutionseries.DNAtemplatesshouldbeusedinamountslessthan100ng.WhenthecDNA(reversetranscriptionsolution)isusedasatemplate,itshouldbeaddedinavolumethatislessthan10%ofthetotalvolumeofthePCRreactionmixture.
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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
ShuttlePCRStandardProtocolStage1:InitialdenaturationReps:195℃ 30sec 20℃/sec
Stage2:PCRReps:4095℃ 5sec 20℃/sec60℃ 20sec 20℃/sec
2. AftergentlycentrifugingLightCyclercapillaries,placethecapillariesintheLightCyclerinstrumentandstartthereaction.TheshuttlePCRstandardprotocolisrecommended.Trythisprotocolfirst,thenoptimizethereactionconditionifneeded.(Referto“GeneralOverviewofPCRConditions”onpage13.)
Note: ThisproductcombinesthehighperformanceofTaKaRaExTaqHS,whichisanenzymeforhotstartPCRthatusesaTaqantibody.InitialdenaturationpriortoPCRshouldbeat95℃for30sec.Thereisnoneedforincubationat95℃for(5-)15minastheinitialdenaturation,suchasrequiredforchemicallymodifiedTaqpolymerases.Excessiveheattreatmentcandecreaseenzymeactivity,andtheamplificationefficiencyandaccuracyinquantificationcanalsobeaffected.
3. Afterreactioncompletion,verifytheamplificationcurves.Establishthestandardcurvewhenabsolutequantificationisperformed.
Refertotheinstructionmanualforthereal-timePCRinstrumentusedforspecificanalysismethods.
9
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
3. Protocol for the Smart Cycler II System
1. PreparethefollowingPCRreactionmixture.
<Perreaction>Reagent Volume Finalconc.PremixExTaq(2X) 12.5μl 1XPCRForwardPrimer(10μM) 0.5μl 0.2μM*1PCRReversePrimer(10μM) 0.5μl 0.2μM*1Probe*2 1μltemplate(<100ng)*3 2μlSterilepurifiedwater 8.5μlTotal 25μl
*1 Thefinalconcentrationofprimersshouldbe0.2μMformostreactions.Ifthisdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
*2 Theprobeconcentrationwilldifferdependingonthespecificreal-timePCRinstrumentandthetypeoffluorescentlabel.Theamountnecessaryshouldbedeterminedbyreferringtotheoperationmanualofinstrumentandtheproductinsertsuppliedwithprobe.Ingeneral,fortheSmartCyclerSystem,thefinalprobeconcentrationshouldbebetween0.1and0.5μM.
*3 ThefinaltemplateconcentrationvariesdependingonthecopynumberoftargetDNApresentinthetemplatesolution.Theoptimalamountshouldbedeterminedbypreparingadilutionseries.DNAtemplatesshouldbeusedinamountslessthan100ng.WhenthecDNA(reversetranscriptionsolution)isusedasatemplate,itshouldbeaddedinavolumethatislessthan10%ofthetotalvolumeofthePCRreactionmixture.
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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
2. GentlycentrifugethereactiontubesusingthecentrifugeexclusivelyforSmartCycler.LoadthereactiontubesintheSmartCyclerIISystemandstartthereaction.TheshuttlePCRstandardprotocolisrecommended.Trythisprotocolfirst,thenoptimizethereactionconditionifneeded.(Referto“GeneralOverviewofPCRConditions”onpage13.)
Stage1:InitialDenaturation Stage2:PCRreaction
ShuttlePCRStandardProtocolStage1:InitialdenaturationHold95℃ 30sec
Stage2:PCRRepeats:4095℃ 5sec60℃ 20sec
Note: ThisproductcombinesthehighperformanceofTaKaRaExTaqHS,whichisanenzymeforhotstartPCRthatusesaTaqantibody.InitialdenaturationpriortoPCRshouldbeat95℃for30sec.Thereisnoneedforincubationat95℃for(5-)15minastheinitialdenaturation,suchasrequiredforchemicallymodifiedTaqpolymerases.Excessiveheattreatmentcandecreaseenzymeactivity,andtheamplificationefficiencyandaccuracyinquantificationcanalsobeaffected.
3. Afterreactioncompletion,verifytheamplificationcurves.Establishastandardcurvewhenabsolutequantificationisdone.
Refertotheinstructionmanualforthereal-timePCRinstrumentusedforspecificanalysismethods.
11
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
4. Protocol for the Mx3000P System
1. PreparethefollowingPCRreactionmixture.<Perreaction>Reagent Volume Finalconc.PremixExTaq(2X) 12.5μl 1XPCRForwardPrimer(10μM) 0.5μl 0.2μM*1PCRReversePrimer(10μM) 0.5μl 0.2μM*1Probe*2 1μlROXReferenceDyeII(50X)*3 0.5μl 1Xtemplate(<100ng)*4 2μlSterilepurifiedwater 8.0μlTotal 25μl
*1 Thefinalconcentrationofprimersshouldbe0.2μMformostreactions.Ifthisdoesnotwork,determinetheoptimalconcentrationswithintherange0.1-1.0μM.
*2 Theprobeconcentrationwilldifferdependingonthespecificreal-timePCRinstrumentandthetypeoffluorescentlabel.Theamountnecessaryshouldbedeterminedbyreferringtotheoperationmanualofinstrumentandtheproductinsertsuppliedwithprobe.
*3 ForMx3000P,ROXReferenceDyeIIisrecommended.*4 Thefinaltemplateconcentrationvariesdependingonthecopynumberof
targetDNApresentinthetemplatesolution.Theoptimalamountshouldbedeterminedbypreparingadilutionseries.DNAtemplatesshouldbeusedinamountslessthan100ng.WhenthecDNA(reversetranscriptionsolution)isusedasatemplate,itshouldbeaddedinavolumethatislessthan10%ofthetotalvolumeofthePCRreactionmixture.
2. Startthereaction. TheshuttlePCRstandardprotocolisrecommended.Trythisprotocolfirst,thenoptimizethereactionconditionsifneeded.(Referto“GeneralOverviewofPCRConditions”onpage13)
ShuttlePCRStandardProtocolStage1:InitialdenaturationReps:195℃ 30sec
Stage2:PCRReps:4095℃ 5sec60℃ 20sec
Note: ThisproductcombinesthehighperformanceofTaKaRaExTasqHS,whichisanenzymeforhotstartPCRthatusesaTaqantibody.InitialdenaturationpriortoPCRshouldbeat95℃for30sec.Thereisnoneedforincubationat95℃for(5-)15minastheinitialdenaturation,suchasrequiredforchemicallymodifiedTaqpolymerases.Excessiveheattreatmentcandecreaseenzymeactivity,andtheamplificationefficiencyandaccuracyinquantificationcanalsobeaffected.
3. Afterreactioncompletion,verifytheamplificationcurves.Establishastandardcurvewhenabsolutequantificationisdone.
Refertotheinstructionmanualforthereal-timePCRinstrumentusedforspecificanalysismethods.
12
Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
5. General Overview of PCR Conditions
InitialdenaturationStep Temperature Time Detection Comment
Initialdenaturation 95℃ 30sec Off
Inmostcases,initialdenaturationat95℃for30secissufficientevenfordifficulttodenaturetemplatessuchascircularplasmidsandgenomicDNAs.Thisproceduremaybeextendedto1-2minat95℃dependingontemplatecondition.Prolongeddenaturationmayinactivatetheenzyme.Therefore,donotperformdenaturationformorethan2min.
ShuttlePCR(2stepPCR) numberofcycles:30-45cyclesStep Temperature Time Detection Comment
Denaturation 95℃ 3-5sec Off
Ingeneral,real-timePCRamplificationproductsdonotexceed300bp.Therefore,denaturationat95℃forabout3-5secisusuallysufficient.
Annealing/extension 56-64℃ 20-30sec
(31,34sec)* On
PleasetrytheStandardProtocol(shuttlePCR,2-stepPCR)first.Whenoptimizingreactionconditions,evaluateresultsusinganannealing/extensiontemperaturebetween56℃and64℃.Ifpoorreactivityisobserved,increasingincubationtimeforthisstepmayimproveresults.
*Someinstrumentsdonotallowadetection-stepsettingoflessthan30sec.AppliedBiosystems7300allowsasettingof31secorlonger.AppliedBiosystems7500allowsasettingof34secorlonger.
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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
VIII. Related ProductsPremixExTaq™(ProbeqPCR)(Cat.#RR390A/B)PrimeScript™RTreagentKit(PerfectRealTime)(Cat.#RR037A/B)PrimeScript™RTreagentKitwithgDNAEraser(PerfectRealTime)(Cat.#RR047A/B)PrimeScript™RTMasterMix(PerfectRealTime)(Cat.#RR036A/B)OneStepPrimeScript™RT-PCRKit(PerfectRealTime)(Cat.#RR064A/B)TBGreen®PremixExTaq™II(TliRNaseHPlus)(Cat.#RR820A/B)TBGreen®PremixExTaq™(TliRNaseHPlus)(Cat.#RR420A/B)OneStepTBGreen®PrimeScript™RT-PCRKitII(PerfectRealTime)(Cat.#RR086A)ThermalCyclerDice™RealTimeSystemIII(Cat.#TP950/TP970/TP980/TP990)*
* Notavailableinallgeographiclocations.Checkforavailabilityinyourarea.
TaKaRaExTaqandTBGreenareregisteredtrademarksofTakaraBioInc.PremixExTaq,ThermalCyclerDice,andPrimeScriptaretrademarksofTakaraBioInc.
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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A
v202202Da
NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTakaraBioInc.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656972orfromourwebsiteatwww.takarabio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.