CLONTECH公司酵母单杂交试剂盒说明书

Matchmaker®GoldYeastOne-HybridLibraryScreeningSystemUserManualCat.Nos.630491,630466,630499PT4087-1PublishedNovember2012UnitedStates/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]®GoldYeastOne-HybridLibraryScreeningSystemUserManualProtocolNo.PT4087-1:SynthesizingandCloningYourpBait-AbAiPlasmid.........................................................10B.Protocol:GeneratingtheBait-ReporterYeastStrains........................................................................11VII.TestingYourBaitStrainforAbArExpression......................................................................13A.Protocol:DeterminingtheMinimalInhibitoryConcentrationofAureobasidinAforYourBait.........13VIII.GeneratingcDNAforLibraryConstruction........................................................................14A.Protocol:FirstStrandSMARTcDNASynthesis...............................................................................15B.Protocol:AmplifyingSMARTcDNAbyLongDistancePCR(LD-PCR).........................................16C.Protocol:PurifyingthedscDNAwithCHROMASPIN+TE-400Columns....................................17IX.CreatingandScreeningaOne-HybridLibrary...................................................................19A.Protocol:CreatingandScreeningaOne-HybridcDNALibrary.......................................................19X.AnalysisofResults...............................................................................................................21XI.ConfirmingPositiveInteractions&RescuingthePreyPlasmid.......................................22A.Protocol:ConfirmationofReporterPhenotypebyRestreaking.........................................................22B.Protocol:YeastColonyPCRAnalysistoEliminateDuplicateClones................................................23C.RescueandIsolationofLibraryPlasmidsResponsibleforReporterActivation..................................23D.Protocol:DistinguishingGenuinePositivefromFalsePositiveInteractions.......................................24E.SequenceAnalysisofaGenuinePositive...........................................................................................26XII.TroubleshootingGuide........................................................................................................27XIII.References...........................................................................................