Premix Ex Taq™ - Takara Bio

Cat. # RR039A

Product Manual

Premix Ex Taq™ (Perfect Real Time)

For Research Use

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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

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Premix Ex Taq™ (Perfect Real Time)Cat. #RR039A

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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.

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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

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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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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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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.

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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



*3 ThefinaltemplateconcentrationvariesdependingonthecopynumberoftargetDNApresentinthetemplatesolution.Theoptimalamountshouldbedeterminedbypreparingadilutionseries.DNAtemplatesshouldbeusedinamountslessthan100ng.WhenthecDNA(reversetranscriptionsolution)isusedasatemplate,itshouldbeaddedinavolumethatislessthan10%ofthetotalvolumeofthePCRreactionmixture.

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ShuttlePCRStandardProtocolStage1:InitialdenaturationReps:195℃ 30sec 20℃/sec

Stage2:PCRReps:4095℃ 5sec 20℃/sec60℃ 20sec 20℃/sec

2. AftergentlycentrifugingLightCyclercapillaries,placethecapillariesintheLightCyclerinstrumentandstartthereaction.TheshuttlePCRstandardprotocolisrecommended.Trythisprotocolfirst,thenoptimizethereactionconditionifneeded.(Referto“GeneralOverviewofPCRConditions”onpage13.)


3. Afterreactioncompletion,verifytheamplificationcurves.Establishthestandardcurvewhenabsolutequantificationisperformed.


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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


*2 Theprobeconcentrationwilldifferdependingonthespecificreal-timePCRinstrumentandthetypeoffluorescentlabel.Theamountnecessaryshouldbedeterminedbyreferringtotheoperationmanualofinstrumentandtheproductinsertsuppliedwithprobe.Ingeneral,fortheSmartCyclerSystem,thefinalprobeconcentrationshouldbebetween0.1and0.5μM.

*3 ThefinaltemplateconcentrationvariesdependingonthecopynumberoftargetDNApresentinthetemplatesolution.Theoptimalamountshouldbedeterminedbypreparingadilutionseries.DNAtemplatesshouldbeusedinamountslessthan100ng.WhenthecDNA(reversetranscriptionsolution)isusedasatemplate,itshouldbeaddedinavolumethatislessthan10%ofthetotalvolumeofthePCRreactionmixture.

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2. GentlycentrifugethereactiontubesusingthecentrifugeexclusivelyforSmartCycler.LoadthereactiontubesintheSmartCyclerIISystemandstartthereaction.TheshuttlePCRstandardprotocolisrecommended.Trythisprotocolfirst,thenoptimizethereactionconditionifneeded.(Referto“GeneralOverviewofPCRConditions”onpage13.)

Stage1:InitialDenaturation Stage2:PCRreaction

ShuttlePCRStandardProtocolStage1:InitialdenaturationHold95℃ 30sec

Stage2:PCRRepeats:4095℃ 5sec60℃ 20sec


3. Afterreactioncompletion,verifytheamplificationcurves.Establishastandardcurvewhenabsolutequantificationisdone.


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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



*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.


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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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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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NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTakaraBioInc.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656972orfromourwebsiteatwww.takarabio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.

Premix Ex Taq™ - Takara Bio

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