Yeast Two-Hybrid System User Manual clontech公司酵母双杂

Matchmaker™GoldYeastTwo-HybridSystemUserManualPT4084-1(PR033493)Cat.No.630489Published30March2010UnitedStates/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]™GoldYeastTwo-HybridSystemUserManualProtocolNo.PT4084-1:ControlMatingProtocol...................................................................................................11VII.CloningandTestingBaitforAutoactivationandToxicity...................................................14A.GenerateaBaitClone.......................................................................................................................14B.DetectingBaitExpression.................................................................................................................15C.Protocol:TestingYourBaitforAutoactivation...................................................................................15D.Protocol:TestingYourBaitforToxicity.............................................................................................16VIII.Two-HybridLibraryScreeningUsingYeastMating...........................................................17IX.AnalysisofResults.................................................................................................................21A.TooFewPositives.............................................................................................................................21B.TooManyPositives..........................................................................................................................21X.ConfirmationofPositiveInteractions&RescueofthePreyPlasmid................................22A.Protocol:YeastColonyPCRAnalysistoEliminateDuplicateClones................................................23B.Protocol:RescueandIsolationofLibraryPlasmidResponsibleforActivationofReporters...............23C.Protocol:DistinguishingGenuinePositivefromFalsePositiveInteractions.......................................24D.SequenceAnalysisofaGenuinePositive...........................................................................................26E.BiochemicalMethodstoConfirmPositiveInteractions.....................................................................27F.DownstreamAnalysis........................................................................................................................27XI.TroubleshootingGuide...........................................................................................................28XII.References.............................................................................................................................30AppendixA:Mate&Plate™LibraryConstruction...................................................................32AppendixB:LibraryTitering.......................................................................................................35AppendixC:ControlPlasmidInformation.................................................................................36AppendixD:YeastGrowthMedia&Supplements...................................................................37TableofContentsCustomerService/Orderingtel:800.662.2566(toll-free)fax:[phone](toll-free)web:@clontech.comTechnicalSupporttel:800.662.2566(toll-free)fax:[phone]web:@clontech.comClontechLaboratories,Inc.–1ATakaraBioCompanyVersionNo.PR0334933Matchmaker™GoldYeastTwo-HybridSystemUserManualTableofContentscontinuedListofFiguresFigure1.Thetwo-hybridprinciple............................................................................................................4Figure2.ReportergeneconstructsinMatchmakeryeaststrains................................................................5Figure3.Two-hybridscreeningusingMate&Plate™Libraries...................................