Takara-PrimeScriptTM-RT-reagent-Kit

TAKARABIOINC.PrimeScriptTMRTreagentKit(PerfectRealTime)#RR037Av.060:Real-timePCR............................................................................4VII.Appendix....................................................................................................6VIII.RelatedProducts.......................................................................................92TAKARABIOINC.PrimeScriptTMRTreagentKit(PerfectRealTime)#RR037Av.060®PremixExTaqTM(PerfectRealTime)orPremixExTaqTM(PerfectRealTime)for2stepRealTimeRT-PCR..5XPrimeScriptTMBuffer(forRealTime)*400μl2.PrimeScriptTMRTEnzymeMixI100μl3.OligodTPrimer50μM100μl4.Random6mers100μM100μl5.RNaseFreedH2Oml6.EASYDilutionBuffer(forRealTimePCR)*2ml*1:containsdNTPMixtureandMg2+.*2:FormeasureddilutionoftotalRNAorcDNAusedasdilutedsolution.IncontrasttodilutionwithwaterorTE,EASYDilutionSolutionfacilitiesaccuratelowconcentrationdilution.DilutedtemplatereagentcanbeappliedasthetemplateforreversetranscriptionorPCRreactions,becauseEasyDilutionSolutiondoesnotinhibiteitherreversetranscriptionorPCRenzymeactivity.EASYDilutionSolutionisalsoavailableseparately.EASYDilution(forRealTimePCR)(TaKaRaCat.#9160)Note:EASYDilutionhasbeentestedwithTaKaRa’sBioreal-timePCRreagent.Compatibilitywithproductsfromothermanufacturershasnotyetbeenverified.Reagentsandinstrumentsnotsuppliedinthiskit.ThermalCycler(or37°C,42°CWaterBath,85°Cheatblock)2.0.2mland.5mlmicrotube(forreversetranscription)3.Micropipettesandpipettetips(autoclaved)-20℃(1)Makesfast,efficientsynthesisofcDNAtemplatesforRealTimePCRpossible.Thiskitisbestsuitedfortwostepreal-timeRT-PCR.(2)ThekitincludesRandom6mersandOligodTPrimerforuseasreversetranscriptionprimers.Thereactioncanbeperformedusingmixturethesetwoprimers,ortheprimercanbeselectedbasedonthepurposeoftheexperiment.Furthermore,GeneSpecificPrimerscanbeusedforspecificgenedetection.(3)AstandardcurvemustbegeneratedforthequantitationofReal-TimeRT-PCR.DilutionoftotalRNAorcDNAafterreversetranscriptionisnecessarybecauselowconcentrationsarerequiredforaviablestandardcurve.However,dilutionwithwaterorTEcannarrowtherangeofthecurveduetounstabledilutionatlowconcentrations.UsingEASYDilutionSolution(forRealTimePCR),fordilutioncausestheresultstobeaccurateatlowerconcentrationsandfacilitatescreationofawide-rangestandardcurve.II.Storage:III.Principle:3TAKARABIOINC.PrimeScriptTMRTreagentKit(PerfectRealTime)#RR037Av.060(1)Itisconvenienttoprepareamastermixofreagentscontaining(RNaseFreedH2O,buffer,enzymes,etc).Usingsuchamixtureallowsaccuratedispensingofreagents,minimizespipettinglosses,andavoidsrepeateddispensingofeachreagent.Thishelpstominimizeexperimentalvariability.(2)GentlyspindownthePrimeScriptTMRTEnzymeMixIpriortopipetting.Pipetenzymesslowlyandcarefullybecauseoftheviscosityofthe50%glycerolinthissolution.(3)Usenewdisposablepipettetipstoavoidcontaminationbetweensampleswhentransferringreagents.(ReferVII.B.RNAsamplepreparation(page8)forRNApreparation).Preparethefollowingreactionmixtureonice.Prepareaslightlylargeramountofmastermixthanisrequiredtocompensateforpipettinglosses.Afterdispensingaliquotsofthismixintothemicrotubes,addtheRNAsample.For1reactionReagentAmountFinalconcentration5×PrimeScriptTMBuffer(forRealTime)2μl1×PrimeScriptTMRTEnzymeMixI0.5μlOligodTPrimer(50μM)*0.5μl25pmolRandom6mers(100μM)*0.5μl50pmoltotalRNARNaseFreedH2OTotal10μl*2*1.UsingbothOligodTPrimerandRandom6mers,efficientsynthesisofcDNAfromtotalmRNAcanbeaccomplished.TherequiredamountofprimerforexclusiveuseofeachprimeroraGeneSpecificPrimerisasfollows.PrimerAmountTotalAmount(pmol)OligodTPrimers(50μM)0.5μl25pmolRandom6mers(100μM)0.5μl50pmolGeneSpecificPrimer(2μM)0.5μl1pmol*2:ItispossibletoscaleuptheRTreactionasneeded.Upto500ngoftotalRNAcanbereversetranscribedin10μlofthereactionmixture.2.Incubatethereactionmixtureunderthefollowingcondition.37℃,5minutes*3(Reversetranscription)85℃,5sec(Inactivationofreversetranscriptasewithheattreatment)4℃V.