赛默飞taq酶说明书 Thermo Scientific

PRODUCTINFORMATIONTaqDNAPolymerase(recombinant#EP0402500ULot:__ExpiryDate:__Concentration:5U/µLStoreat-20°COrderingInformationTaqDNAPolymerase(recombinantComponent#EP0401#EP0402#EP0405#EP0406TaqDNAPolymerase,5U/µL100U500U5x500U10x500U10XTaqBufferwithKCl0.6mL2x1.25mL10x1.25mL20x1.25mL10XTaqBufferwith(NH42SO40.6mL2x1.25mL10x1.25mL20x1.25mL25mMMgCl20.6mL2x1.25mL10x1.25mL20x1.25mLTaqDNAPolymerase(recombinant,LCComponent#EP0403#EP0404TaqDNAPolymerase,1U/µL100U500U10XTaqBufferwithKCl0.6mL2x1.25mL10XTaqBufferwith(NH42SO40.6mL2x1.25mL25mMMgCl20.6mL2x1.25mLRev.12VDescriptionTaqDNAPolymeraseisahighlythermostableDNApolymeraseofthethermophilicbacteriumThermusaquaticus.Theenzymecatalyzes5’→3’synthesisofDNA,hasnodetectable3’→5’exonuclease(proofreadingactivityandpossesses5’→3’exonucleaseactivity.Inaddition,TaqDNAPolymeraseexhibitsdeoxynucleotidyltransferaseactivity,whichfrequentlyresultsintheadditionofextraadeninesatthe3’-endofPCRproducts.RecombinantTaqDNAPolymeraseisidealforstandardPCRofamplicons5kborshorter.Applications•RoutinePCRamplificationofDNAfragmentsupto5kb(1.•GenerationofPCRproductforTAcloning.•DNAlabeling(2-4.•DNAsequencing(5.SourceE.colicellswithaclonedpolgenefromThermusaquaticusYT1.DefinitionofActivityUnitOneunitoftheenzymecatalyzestheincorporationof10nmolofdeoxyribonucleotidesintoapolynucleotidefraction(adsorbedonDE-81in30minat70°C.Enzymeactivityisassayedinthefollowingmixture:67mMTris-HCl(pH8.8at25°C,6.7mMMgCl2,1mM2-mercaptoethanol,50mMNaCl,0.1mg/mLBSA,0.75mMactivatedsalmonmiltDNA,0.2mMofeachdNTP,0.4MBq/mL[3H]-dTTP.StorageBufferTheenzymeissuppliedin:20mMTris-HCl(pH8.0,1mMDTT,0.1mMEDTA,100mMKCl,0.5%(v/vNonidet®P40,0.5%(v/vTween®20and50%(v/vglycerol.10XTaqBufferwithKCl100mMTris-HCl(pH8.8at25°C,500mMKCl,0.8%(v/vNonidetP40.10XTaqBufferwith(NH42SO4750mMTris-HCl(pH8.8at25°C,200mM(NH42SO4,0.1%(v/vTween20.InhibitionandInactivation•Inhibitors:ionicdetergents(deoxycholate,sarkosylandSDSatconcentrationshigherthan0.06,0.02and0.01%,respectively(6.•Inactivatedbyphenol/chloroformextraction.PROTOCOLToprepareseveralparallelreactionsandtominimizethepossibilityofpipettingerrors,prepareaPCRmastermixbymixingwater,buffer,dNTPs,primersandTaqDNAPolymerase.Preparesufficientmastermixforthenumberofreactionsplusoneextra.AliquotthemastermixintoindividualPCRtubesandthenaddtemplateDNA.1.Gentlyvortexandbrieflycentrifugeallsolutionsafterthawing.2.Placeathin-walledPCRtubeoniceandaddthefollowingcomponentsforeach50µLreaction:10XTaqBuffer5µLdNTPMix,2mMeach(#R02415µL(0.2mMofeachForwardprimer0.1-1.0µMReverseprimer0.1-1.0µM25mMMgCl2*1-4mMTemplateDNA10pg-1µgTaqDNAPolymerase1.25UWater,nuclease-free(#R0581to50µLTotalvolume50µL*Volumesof25mMMgCl2,requiredforspecificfinalMgCl2concentration:FinalconcentrationofMgCl2,mM11.522.534Volumeof25mMMgCl2tobeaddedfor50µLreaction,µL2345683.Gentlyvortexthesamplesandspindown.4.Ifusingathermalcyclerthatdoesnotuseaheatedlid,overlaythereactionmixturewith25µLofmineraloil.5.PerformPCRusingrecommendedthermalcyclingconditions:Step°CTimeNumberofcyclesInitialdenaturation951-3min1Denaturation9530s25-40AnnealingTm-530sExtension721min/kbFinalExtension725-15min1GUIDELINESFORPREVENTINGCONTAMINATIONOFPCRREACTIONDuringPCRmorethan10millioncopiesoftemplateDNAaregenerated.Therefore,caremustbetakentoavoidcontaminationwithothertemplatesandampliconsthatmaybepresentinthelaboratoryenvironment.Generalrecommendationstolowertheriskofcontaminationareasfollows:•PrepareyourDNAsample,setupthePCRmixture,performthermalcyclingandanalyzePCRproductsinseparateareas.•SetupPCRmixturesinalaminarflowcabinetequippedwithanUVlamp.•WearfreshglovesforDNApurificationandreactionsetup.•UsereagentcontainersdedicatedforPCR.Usepositivedisplacementpipettes,orusepipettetipswithaerosolfilterstoprepareDNAsamplesandperformPCRsetup.•UsePCR-certifiedreagents,includinghighqualitywater(e.g.,Water,nuclease-free,(#R0581.•Alwaysperform“notemplatecontrol”(NTCreactionstocheckforcontamination.GUIDELINESFORPRIMERDESIGNUsetheThermoScientificREviewerprimerdesignsoftwarerecommendationsforPCRprimerdesignasoutlinedbelow:•PCRprimersaregenerally15-30nucleotideslong.•OptimalGCcontentoftheprimeris40-60%.Ideally,CandGnucleotidesshouldbedistributeduniformlyalongtheprimer.•AvoidplacingmorethanthreeGorCnucleotidesatthe3’-endtolowertheriskofnon-specificpriming.•Ifpossible,theprimershouldterminatewithaGorCatthe3’-end.•Avoidself-complementaryprimerregions,complementaritiesbetweentheprimersanddirectprimerrepeatstopreventhairpinformationandprimerdimerization.•CheckforpossiblesitesofundesiredcomplementarybetweenprimersandtemplateDNA.•Whendesigningdegenerateprimers,placeatleast3conservatednucleotidesatthe3’-end.•Whenintroducingrestrictionenzymesitesintoprimers,refertothetable“CleavageefficiencyclosetotheterminiofPCRfragments”locatedonnumberofextrabasesrequiredforefficientcleavage.•Differencesinmeltingtemperatures(Tmbetweenthetwoprimersshouldnotexceed5°C.(continuedonreversepageEstimationofprimermeltingtemperatureForprimerscontaininglessthan25nucleotides,theapprox.meltingtemperature(Tmcanbecalculatedusingthefollowingequation:Tm=4(G+C+2(A+T,whereG,C,A,Trepresentthenumberofrespectivenucleotidesintheprimer.Iftheprimercontainsmorethan25nucleotidesspecializedcomputerprogramse.g.,REviewer™accountforinteractionsofadjacentbases,effectofsaltconcentration,etc.COMPONENTSOFTHEREACTIONMIXTURETemplateDNAOptimalamountsoftemplateDNAinthe50µLreactionvolumeare0.01-1ngforbothplasmidandphageDNA,and0.1-1µgforgenomicDNA.Higheramountoftemplateincreasestheriskofgenerationofnon-specificPCRproducts.Loweramountoftemplatereducestheaccuracyoftheamplification.AllroutineDNApurificationmethodsaresuitablefortemplatepreparatione.g.,ThermoScientificGeneJETGenomicDNAPurificationKit(#K0721orGeneJET™PlasmidMiniprepKit(#K0502.TraceamountsofcertainagentsusedforDNApurification,suchasphenol,EDTAandproteinaseK,caninhibitDNApolymerases.EthanolprecipitationandrepeatedwashesoftheDNApelletwith70%ethanolnormallyremovestracecontaminantsfromDNAsamples.MgCl2concentrationDuetothebindingofMg2+todNTPs,primersandDNAtemplates,Mg2+concentrationneedstobeoptimizedformaximalPCRyield.Therecommendedconcentrationrangeis1-4mM.IftheMg2+concentrationistoolow,theyieldofPCRproductcouldbereduced.Onthecontrary,non-specificPCRproductsmayappearandthePCRfidelitymaybereducediftheMg2+concentrationistoohigh.IftheDNAsamplescontainEDTAorothermetalchelators,theMg2+ionconcentrationinthePCRmixtureshouldbeincreasedaccordingly(1moleculeofEDTAbindsoneMg2+.dNTPsTherecommendedfinalconcentrationofeachdNTPis0.2mM.IncertainPCRapplications,higherdNTPconcentrationsmaybenecessary.DuetothebindingofMg2+todNTPs,theMgCl2concentrationneedstobeadjustedaccordingly.Itisessentialtohaveequalconcentrationsofallfournucleotides(dATP,dCTP,dGTPanddTTPpresentinthereactionmixture.Toachieve0.2mMconcentrationofeachdNTPinthePCRmixture,usethefollowingvolumesofdNTPmixes:VolumeofdNTPMix,2mMeach(#R0241dNTPMix,10mMeach(#R0191dNTPMix,25mMeach(#R112150µL5µL1µL0.4µL25µL2.5µL0.5µL0.2µL20µL2µL0.4µL0.16µLPrimersTherecommendedconcentrationrangeofthePCRprimersis0.1-1µM.Excessiveprimerconcentrationsincreasetheprobabilityofmisprimingandgenerationofnon-specificPCRproducts.Fordegenerateprimershigherprimerconcentrationsintherangeof0.3-1µMareoftenfavorable.CYCLINGPARAMETERSInitialDNAdenaturationItisessentialtocompletelydenaturethetemplateDNAatthebeginningofPCRtoensureefficientutilizationofthetemplateduringthefirstamplificationcycle.IftheGCcontentofthetemplateis50%orless,aninitial1-3mindenaturationat95°Cissufficient.ForGC-richtemplatesthisstepshouldbeprolongedupto10min.Iflongerinitialdenaturationstepisrequired,ortheDNAisdenaturedatahighertemperature,TaqDNAPolymeraseshouldbeaddedaftertheinitialdenaturationsteptoavoidadecreaseinitsactivity.DenaturationADNAdenaturationtimeof30secondspercycleat95°Cisnormallysufficient.ForGC-richDNAtemplates,thisstepcanbeprolongedto3-4min.DNAdenaturationcanalsobeenhancedbytheadditionofeither10-15%glycerolor10%DMSO,5%formamideor1-1.5Mbetaine.Themeltingtemperatureoftheprimer-templatecomplexdecreasessignificantlyinthepresenceofthesereagents.Therefore,theannealingtemperaturehastobeadjustedaccordingly.Inaddition,10%DMSOand5%formamideinhibitDNApolymerasesby50%.Thus,theamountoftheenzymeshouldbeincreasediftheseadditivesareused.PrimerannealingTheannealingtemperatureshouldbe5°Clowerthanthemeltingtemperature(Tmoftheprimers.Annealingfor30secondsisnormallysufficient.Ifnon-specificPCRproductsappear,theannealingtemperatureshouldbeoptimizedstepwisein1-2°Cincrements.Whenadditiveswhichchangethemeltingtemperatureoftheprimer-templatecomplexareused(glycerol,DMSO,formamideandbetaine,theannealingtemperaturemustalsobeadjusted.ExtensionTheoptimalextensiontemperatureforTaqDNAPolymeraseis70-75°C.Therecommendedextensionstepis1minat72°CforPCRproductsupto2kb.Forlargerproducts,theextensiontimeshouldbeprolongedby1min/kb.NumberofcyclesThenumberofcyclesmayvarydependingontheamountoftemplateDNAinthePCRmixtureandtheexpectedPCRproductyield.Iflessthan10copiesofthetemplatearepresentinthereaction,about40cyclesarerequired.Forhighertemplateamounts,25-35cyclesaresufficient.FinalextensionAfterthelastcycle,itisrecommendedtoincubatethePCRmixtureat72°Cforadditional5-15mintofill-inanypossibleincompletereactionproducts.IfthePCRproductwillbeclonedintoTAvectors(forinstance,usingThermoScientificInsTAclonePCRCloningKit(#K1213,thefinalextensionstepmaybeprolongedto30mintoensurethehighestefficiencyof3’-dAtailingofPCRproduct.IfthePCRproductwillbeusedforcloningusingThermoScientificCloneJETPCRCloningKit(#K1231,thefinalextensionstepcanbeomitted.TroubleshootingFortroubleshootingpleasevisitReferences1.Innis,M.A.,etal.,PCRProtocolsandApplications:ALaboratoryManual,Academic,NewYork,1989.2.Celeda,D.,etal.,PCRamplificationandsimultaneousdigoxigeninincorporationoflongDNAprobesforfluorescenceinsituhybridization,BioTechniques,12,98-102,1992.3.Finckh,U.,etal.,Producingsingle-strandedDNAprobeswiththeTaqDNApolymerase:ahighyieldprotocol,BioTechniques,10,35-39,1991.4.Yu,H.etal.,CyaninedyedUTPanalogsforenzymaticlabelingofDNAprobes,NucleicAcidsRes.,22,3226-3232,1994.5.Innis,M.A.,etal.,DNAsequencingwithThermusaquaticusDNApolymeraseanddirectsequencingofpolymerasechainreaction-amplifiedDNA,Proc.Natl.Acad.Sci.USA,85,9436-9440,1988.6.Weyant,R.S.,etal.,EffectofionicandnonionicdetergentsontheTaqpolymerase,Biotechniques,9,309-308,1990.7.Lundberg,K.S.,etal.,High-fidelityamplificationusingathermostableDNApolymeraseisolatedfromPyrococcusfuriosus,Gene,108,1-6,1991.CERTIFICATEOFANALYSISEndodeoxyribonucleaseAssayNoconversionofcovalentlyclosedcircularDNAtonickedDNAwasdetectedafterincubationof10UofTaqDNAPolymerasewith1µgofpUC19DNAfor4hoursat37°C.ExodeoxyribonucleaseAssayNodegradationofDNAwasobservedafterincubationof1µgoflambdaDNA/HindIIIfragmentswith10UTaqDNAPolymerasefor4hoursat37°C.RibonucleaseAssayNocontaminatingRNaseactivitywasdetectedafterincubationof10UofTaqDNAPolymerasewith1µgof[3H]-RNAfor4hoursat37°C.FunctionalAssayTaqDNAPolymerasewastestedforamplificationof950bpsinglecopygenefromhumangenomicDNAandforamplificationofcDNA.Qual