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SMART™cDNALibraryConstructionKitUserManualCat.No.634901PT3000-1(PR043527)PublishedMay2010UnitedStates/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]™cDNALibraryConstructionKitUserManualI.Introduction4II.ListofComponents9III.AdditionalMaterialsRequired12IV.GeneralConsiderations15V.SMARTcDNASynthesisbyLDPCR18A.First-StrandcDNASynthesis18B.cDNAAmplificationbyLDPCR19C.ProteinaseKDigestion21D.SfiIDigestion21E.cDNASizeFractionationbyCHROMASPIN-40022VI.SMARTcDNASynthesisbyPrimerExtension24A.First-StrandcDNASynthesis24B.dscDNASynthesisbyPrimerExtension25C.ProteinaseKDigestion26D.SfiIDigestion27E.cDNASizeFractionationbyCHROMASPIN-40027VII.SMARTcDNALibraryProtocols30A.LigationofcDNAtoλTriplEx2Vector30B.BacterialCulturePlating31C.TiteringtheUnamplifiedLibrary32D.DeterminingthePercentageofRecombinantClones33E.LibraryAmplification34F.TiteringtheAmplifiedLibrary35VIII.TroubleshootingGuide37IX.References42AppendixA:TypicalResultsofdscDNASynthesis43AppendixB:ConvertingλTriplEx2topTriplEx245AppendixC:RestrictionMapofλTriplEx248TableofContentsProtocolNo.PT3000-1™cDNALibraryConstructionKitUserManualTableI.Bacterialhoststraingenotypes11TableII.Hoststrainapplications&mediaadditives11TableIII.RelationshipbetweenamountofRNAstartingmaterial&optimalnumberofthermalcycles20TableIV.LigationsusingthreedifferentratiosofcDNAtophagevector30TableV.Platingdilutionsfortiteringanamplifiedlibrary36ListofFiguresFigure1.FlowchartoftheSMARTcDNALibraryConstructionKitprotocols5Figure2.ComparisonofSfiI(A&B)recognitionsequences7Figure3.GenerationofpolypeptidesfromallthreereadingframesinasinglerecombinantλTriplEx2clone8Figure4.GuidetousingtheSMARTcDNALibraryConstructionKitprotocols17Figure5.GuidetotroubleshootingSMARTcDNAsynthesis37Figure6.TypicalResults:dscDNAsynthesizedusingtheSMARTcontrolreagents&protocols43Figure7.ConversionofrecombinantλTriplEx2tothecorrespondingpTriplEx247Figure8.RestrictionmapofλTriplEx248ListofTablesContactUsForAssistanceCustomerService/Ordering:TechnicalSupport:Telephone:[phone](toll-free)Telephone:[phone](toll-free)Fax:[phone](toll-free)Fax:[phone]Web::[e-mail]-mail:[e-mail],Inc.™cDNALibraryConstructionKitUserManualTheSMARTTMcDNALibraryConstructionKitprovidesamethodforproduc-inghigh-quality,full-lengthcDNAlibrariesfromnanogramsoftotalorpolyA+RNA.Thiskitcontainstwoseparateprotocols,allowingyoutochooseamethodbasedonyourstartingmaterial.Thefirstprotocolemploysanovel,PCR-basedmethodforresearcherslimitedbytheirstartingmaterial(i.e.,50ngoftotalRNA).Thesecondprotocolprovidesamoretraditionalprotocolforresearcherswithabundantamountsofstartingmaterial(i.e.,1µgormorepolyA+RNA).BothprotocolsutilizethepatentedSMARTIVTM(SwitchingMechanismAt5'endofRNATranscript)Oligonucleotideinthefirst-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’Allessio,1988).TheSMARTprotocolsaredesignedtopreferentiallyenrichforfull-lengthcDNAs,whileeliminatingT4DNApolymeraseandadaptorligation.SMARTlibrariesareproventocontainahigherpercentageoffull-lengthclonesthanlibrariesconstructedbycon-ventionalmethods(October1998Clontechniques)orotherfull-lengthcDNAsynthesisprotocols(Okayama&Berg,1982;Katoetal.,1994).Thus,clonesisolatedfromSMARTcDNAlibrariescontainsequencescorrespondingtothecomplete5'untranslatedregionofthemRNA(ibid.).SMARTTMpreservesthecomplete5'mRNAsequenceLibrariesmadewithSMARTcDNAcanfacilitatepreliminarymappingoftranscriptionstartsites(Fromont-Racine,1993)duetothehighpercentageoffull-lengthclones.However,SMARTcDNAlibrariesmaynotbesuitableforimmunoscreeningforcertainproteins.Insomecases,5'untranslatedregions(UTRs)maycontainstopcodonsinframewiththeinitiatingtransla-tionstartsiteintheexpressionvector.I.IntroductionProtocolNo.PT3000-1™cDNALibraryConstructionKitUserManualI.IntroductioncontinuedFigure1.FlowchartoftheSMARTcDNALibraryConstructionKitprotocols.Theright