clontech公司RACE试剂盒说明书

SMARTer™RACEcDNAAmplificationKitUserManualCat.Nos.634923&634924PT4096-1(PR053541)PublishedJune2010NowincludingRandomPrimers&SMARTScribe™ReverseTranscriptase!UnitedStates/Canada800.662.2566AsiaPacific+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.543.6116ClontechLaboratories,Inc.ATakaraBioCompany1290TerraBellaAve.MountainView,CA94043TechnicalSupport(US)E-mail:[e-mail]™RACEcDNAAmplificationKitUserManualProtocolNo.PT4096-1:First-StrandcDNASynthesis....................................................................................12VI.RapidAmplificationofcDNAEnds(RACE)...........................................................................14A.ThingsYouShouldKnowBeforeStartingRACEPCRReactions......................................................14B.PRoToCoL:PositiveControlRACEPCRExperiment.................................................................14C.ControlPCRReactions....................................................................................................................16D.PRoToCoL:RapidAmplificationofcDNAEnds(RACE)............................................................17VII.CharacterizationofRACEProducts.....................................................................................20A.ComparisonofRACEProductsobtainedwithGSPs&NGSPs......................................................20B.SouthernBlotAnalysis......................................................................................................................20C.PRoToCoL:NucleoTrap®GelExtraction.....................................................................................21D.Cloning&SequencingRACEProducts............................................................................................22VIII.TroubleshootingGuide.........................................................................................................23IX.References..............................................................................................................................27AppendixA:DetailedFlowChartof5’RACE............................................................................28AppendixB:DetailedFlowChartof3’RACE............................................................................29AppendixC:SuppressionPCRandStep-OutPCR....................................................................30AppendixD:5’-RACEcDNAAmplificationwithRandomPrimers..........................................32TableofContentsListofFiguresFigure1.MechanismofSMARTercDNAsynthesis..................................................................................5Figure2.overviewoftheSMARTerRACEprocedure..............................................................................6Figure3.Therelationshipofgene-specificprimerstothecDNAtemplate.................................................8Figure4.5’-and3’-RACEsampleresults................................................................................................16Figure5.Detailedmechanismofthe5’-RACEreactions.........................................................................28Figure6.Detailedmechanismofthe3’-RACEreactions.........................................................................29Figure7.MechanismsofsuppressionPCRandstep-outPCR......................................