taq_说明书

taq_说明书Platinum®TaqDNAPolymeraseCat.Nos.10966-018(100reactions)10966-034(500reactions)10966-026(250reactions)10966-083(5,000reactions)Conc:5U/µLStoreat–20°Cinanon-frost-freefreezerKitContentsSizeKitrxn5,000rxnComponent100rxn250rxn500Platinum®TaqDNAPolymerase20μL50μL100μL1000μL10XPCRBuffer,MinusMg1.25mL1.25mL2.5mL50mL50mMMagnesiumChloride1mL1mL1mL10mLDescriptionPlatinum®TaqDNAPolymeraseisrecombinantTaqDNApolymerasecomplexedwithaproprietaryantibodythatblockspolymeraseactivityatambienttemperatures.ActivityisrestoredafterthedenaturationstepinPCRcyclingat94°C,therebyprovidinganautomatic“hotstart”forTaqDNApolymeraseinPCR(1,2,3).HotstartsinPCRprovideincreasedsensitivity,specificity,andyield,whileallowingassemblyofreactionsatroomtemperature.TheuseofthisantibodyhelpsreducePCRoptimizationrequirements,reactionset-uptimeandeffort,handlingofreactioncomponents,andcontaminationrisk.LikestandardTaqDNAPolymerase,Platinum®Taqhasanontemplate-dependentterminaltransferaseactivitythataddsasingledeoxyadenosine(A)tothe3'endsofPCRproducts.LikestandardTaq,ithasboth5'→3'polymeraseand5'→3'exonucleaseactivity,butlacks3'→5'exonucleaseactivity.Platinum®Taqissuppliedatthesame5unitperμLconcentrationasTaqDNAPolymerase.NomodificationtoPCRreactionsorprotocolsarenecessary.Thisenzymeformulationcanalsobeusedinlargervolumecocktailmixeswithoutdifficulty.Partno:10966.ppsMAN0000925Rev.date:10May2010Fortechnicalsupport,emailtech_support@invitrogen.com.Forcountry-specificcontactinformation,visitwww.invitrogen.com.Page2of4QualityControlTheCertificateofAnalysisprovidesdetailedqualitycontrolinformationforeachproduct.CertificatesofAnalysisareavailableonourwebsite.Gotowww.invitrogen.com/supportandsearchfortheCertificateofAnalysisbyproductlotnumber,whichisprintedonthebox.StorageBuffer20mMTris-HCl(pH8.0),40mMNaCl,2mMSodiumPhosphate,0.1mMEDTA,1mMDTT,stabilizers,50%(v/v)glycerolUnitDefinitionOneunitofPlatinum®TaqDNAPolymeraseincorporates10nmolofdeoxyribonucleotideintoacid-precipitablematerialin30minutesat74°C.GuidelinesandRecommendations•SincePCRisapowerfultechniquecapableofamplifyingtraceamountsofDNA,allappropriateprecautionsshouldbetakentoavoidcross-contamination.•IfthePCRefficiencyisnotoptimal,repeatthereactionwithdifferentprimerconcentrationsfrom100to500nM(finalconcentration),in100-nMincrements.•Aconcentrationof1.5mMMgCl2issufficientformosttargets.Forfurtheroptimization,prepareatitrationfrom1.5mMto3mMin0.25-mMincrements.•ForlongergenomicDNAtargets,werecommendusing2–2.5UofPlatinum®TaqDNAPolymeraseandincreasingtheextensiontimeasspecified(1minuteperkb).Page3of4ProtocolThefollowingprocedureissuggestedasaguidelineandstartingpoint.Optimalreactionconditions(incubationtimesandtemperatures,concentrationofPlatinum®TaqDNAPolymerase,primers,MgCl2,andtemplateDNA)varyandneedtobeoptimized.1.Addthefollowingcomponentstoasterile0.5-mLmicrocentrifugetube.Volumesareforasingle50-μLreaction.Volumescanbescaledasneeded.Prepareamastermixofcommoncomponentsformultiplereactions.ConcentrationVolumeFinalComponents10XPCRBuffer,MinusMg5μL1X10mMdNTPmixture1μL0.2mMeachμL1.5mM50mMMgCl21.5Primermix(10μMeach)1μL0.2μMeachTemplateDNA≥1μL(asrequired)Platinum®TaqDNAPolymerase0.2μL1.0unit*Autoclaved,distilledwaterto50μLNotapplicable*1.0unitissufficientforamplifyingmosttargets.Insomecases,moreenzymemayberequired(upto2.5units).2.Capthetubes,mix,andcentrifugebrieflytocollectthecontents.4.Incubatetubesinathermalcyclerat94°Cfor30secondsto2minutestocompletelydenaturethetemplateandactivatetheenzyme.5.Perform25–35cyclesofPCRamplificationasfollows:Denature94°Cfor30secondsAnneal55°Cfor30secondsExtend72°Cfor1minutesperkb6.Maintainthereactionat4°Caftercycling.Thesamplescanbestoredat–20°Cuntiluse.Analyzetheproductsbyagarosegelelectrophoresis.WerecommendusingE-Gel®1.2%gelsandTrackIt™100bpor1kbPlusDNAladders(seeAdditionalProductsonpage4).Page4of4AdditionalProductsThefollowingproductsareavailableonourwebsiteatwww.invitrogen.comorbycontactingtechnicalsupport.ProductAmountCatalogno.10mMdNTPMix,PCRGrade100μL18427-01310mMdNTPMix,PCRGrade1mL18427-088E-Gel®1.2%StarterPak6gelsplusPowerBase™G6000-01E-Gel®1.2%18-Pak18gelsG5018-01TrackIt™100bpDNALadder100applications10488-058TrackIt™1kbPlusDNALadder100applications10488-085References1.Chou,Q.,etal.(1992)Nucl.AcidsRes.20,1717.2.Sharkey,D.J.,etal.(1994)BioTechnology12,506.3.Westfall,B.A.,etal.(1997)Focus®19.3,46.LimitedUseLabelLicenseNo.1:ThermostablePolymerasesUseofthisproductiscoveredbyoneormoreofthefollowingUSpatentsandcorrespondingpatentclaimsoutsidetheUS:5,789,224,5,618,711,and6,127,155.Thepurchaseofthisproductincludesalimited,non-transferableimmunityfromsuitundertheforegoingpatentclaimsforusingonlythisamountofproductforthepurchaser’sowninternalresearch.Norightunderanyotherpatentclaim,norighttoperformanypatentedmethod,andnorighttoperformcommercialservicesofanykind,includingwithoutlimitationreportingtheresultsofpurchaser'sactivitiesforafeeorothercommercialconsideration,isconveyedexpressly,byimplication,orbyestoppel.Thisproductisforresearchuseonly.DiagnosticusesunderRochepatentsrequireaseparatelicensefromRoche.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.LimitedUseLabelLicenseNo.14:DirectInhibitionbyAnti-PolymeraseAntibodiesLicensedtoInvitrogenCorporation,underU.S.PatentNos.5,338,671;5,587,287,andforeignequivalentsforuseinresearchonly.©2010LifeTechnologiesCorporation.Allrightsreserved.Forresearchuseonly.Notintendedforanyanimalorhumantherapeuticordiagnosticuse.