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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:tech@clontech.com™SMART™cDNALibraryConstructionKitUserManualCat.No.634903PT3577-1(PR932722)Published11March2009ClontechLaboratories,Inc.™SMART™cDNALibraryConstructionKitUserManualI.Introduction4II.ListofComponents10III.AdditionalMaterialsRequired12IV.GeneralConsiderations14V.SMARTcDNASynthesisbyLDPCR17A.First-StrandcDNASynthesis17B.cDNAAmplificationbyLDPCR18C.ProteinaseKDigestion20D.SfiIDigestion21E.cDNASizeFractionation21VI.SMARTcDNASynthesisbyPrimerExtension24A.First-StrandcDNASynthesis24B.dscDNASynthesisbyPrimerExtension25C.ProteinaseKDigestion26D.SfiIDigestion27E.cDNASizeFractionation27VII.LigationofdscDNAtopDNR-LIB30VIII.TransformationofRecombinantPlasmidsintoE.coli31IX.TiteringPlasmidLibraries33X.AmplificationofPlasmidLibraries34XI.TroubleshootingGuide36XII.References41AppendixA:TypicalResultsofPCRanddscDNASynthesis43AppendixB:PreparationofElectrocompetentE.coli45AppendixC:pDNR-LIBVectorMapandMCS46AppendixD:LibraryScreeningbyPCR47TableofContentsContactUsForAssistanceCustomerService/Ordering:TechnicalSupport:Telephone:800.662.2566(toll-free)Telephone:800.662.2566(toll-free)Fax:800.424.1350(toll-free)Fax:650.424.1064Web::orders@clontech.comE-mail:tech@clontech.comProtocolNo.PT3577-1™SMART™cDNALibraryConstructionKitUserManualTableI.RelationshipbetweenamountofRNAstartingmaterialandoptimalnumberofthermalcycles19TableII.LigationsusingthreedifferentratiosofcDNAtovector30ListofFiguresFigure1.FlowchartoftheCreatorSMARTcDNALibraryConstructionKitprotocols5Figure2.CreatorSMARTlibrariesallowyoutotransferyourgeneintomultipleexpressionsystemsquicklyandinexpensively7Figure3.ComparisonoftheSfiI(A&B)recognitionsequences8Figure4.GuidetousingtheCreatorSMARTcDNALibraryConstructionKitprotocols16Figure5.GuidetotroubleshootingSMARTcDNAsynthesis36Figure6.TypicalResults:dscDNAsynthesizedusingtheSMARTcontrolreagents&protocols43Figure7.pDNR-LIBVectorMapandMCS.46ListofTablesClontechLaboratories,Inc.™SMART™cDNALibraryConstructionKitUserManualTheCreatorTMSMARTTMcDNALibraryConstructionKitprovidesadependablemethodforproducinghigh-quality,Creator-compatiblecDNAlibraries.ClontechSMARTtechnologymakesitpossibletogeneratefull-length,directionallyclonedcDNALibrariesfromnanogramsoftotalorpolyA+RNA.UsingtheCreatorCloningSystem,isolatedclonesfromfinishedlibrariescanbetransferreddirectlytoacceptorexpressionvectorsforfunctionalanalysis—withouttheneedforsubcloning.ThisUserManualcontainstwoseparateprotocols,allowingyoutochooseamethodbasedonyourstartingmaterial.Thefirstprotocolemploysanovel,PCR-basedmethodforresearcherslimitedbytheirstartingmaterial(i.e.,50ngoftotalRNA).Thesecondprotocolprovidesamoretraditionalprocedureforresearcherswithabundantamountsofstartingmaterial(i.e.,1µgormorepolyA+RNA).BothprotocolsutilizetheSMARTIVOligonucleotide(patentpending)inthefirst-strandsynthesistogeneratehighyieldsoffull-length,double-stranded(ds)cDNA(Figure1).Full-lengthcDNAwithcomplete5'endsAllcommonlyusedcDNAsynthesismethodsrelyontheabilityofreversetranscriptase(RT)totranscribemRNAintosingle-stranded(ss)DNAinthefirst-strandreaction.Insomecases,RTterminatesbeforetranscribingthecompletemRNAsequence.ThisisparticularlytrueforlongmRNAs,especiallyifthefirst-strandsynthesisisprimedwitholigo(dT)primersonlyorifthemRNAcontainsabundantsecondarystructures.Inaddition,conventionalcDNAcloningproceduresusetheT4DNApolymerasetogeneratebluntcDNAendsaftersecond-strandsynthesis.Asaresult,under-represented5'endsofgenesincDNApopulationstendtobe5–30nucleotidesshorterthantheoriginalmRNA(D’Alessio,1988).TheSMART(SwitchingMechanismAt5'endofRNATranscript)protocolsaredesignedtopreferentiallyenrichforfull-lengthcDNAs,whileeliminatingT4DNApolymeraseandadaptorligation.SMARTlibrariesareproventocontainahigherpercentageoffull-lengthclonesthanlibrariesconstructedbyconventionalmethods(October1998CLONTECHniques)orotherfull-lengthcDNAsynthesisprotocols(Okayama&Berg,1982;Katoetal.,1994).ClonesisolatedfromSMARTcDNAlibrariesareevenfoundtocontainsequencescorrespondingtothecomplete5'untranslatedregionofthemRNA(ibid.).CreatorSystemoverviewTheCreatorSystemallowsthetransferofatargetgenefromasingledonorvectordirectlyintomultipleacceptorexpressionvectorsusingCre-loxPrecombination.Usingthismethod,anygeneclonedintoaspecializedcloningvector(suchaspDNR-LIB)canbetransferredintoanyacceptorvectorforfunctionalanalysiswithouttheneedforsubcloning(Figure2).CrerecombinasemediatesrecombinationbetweenorwithinDNAsequencesatspecificlocationscalledloxPsites(Sauer,1994;Abremskietal.,1984).TheseI.IntroductionProtocolNo.PT3577-1