yeast-protocol-handbook-clontech

YeastProtocolsHandbookFORRESEARCHUSEONLYPT3024-1(PR973283)PublishedJuly2009UnitedStates/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]:Urea/SDSMethod13D.PreparationofProteinExtracts:TCAMethod15E.Troubleshooting17V.YeastTransformationProcedures18A.GeneralInformation18B.ReagentsandMaterialsRequired19C.TipsforaSuccessfulTransformation20D.IntegratingPlasmidsintotheYeastGenome20E.Small-scaleLiAcYeastTransformationProcedure20F.TroubleshootingYeastTransformation22VI.α-andβ-GalactosidaseAssays23A.GeneralInformation23B.InvivoPlateAssayUsingX-galintheMedium26C.Colony-liftFilterAssay26D.LiquidCultureAssayUsingONPGasSubstrate27E.LiquidCultureAssayUsingCPRGasSubstrate28F.LiquidCultureAssayUsingaChemiluminescentSubstrate29G.α-GalQuantitativeAssay32VII.WorkingwithYeastPlasmids34A.GeneralInformation34B.PlasmidIsolationFromYeast34C.TransformingE.coliwithYeastPlasmids36VIII.AnalysisofYeastPlasmidInsertsbyPCR39A.GeneralInformation39B.TipsforSuccessfulPCRofYeastPlasmidTemplates39IX.AdditionalUsefulProtocols42A.YeastColonyHybridization42B.GeneratingYeastPlasmidSegregants43C.YeastMating44X.References46APPENDICESA.GlossaryofTechnicalTerms49B.YeastGeneticMarkersUsedintheMatchmakerSystems51C.MediaRecipes52A.YeastMedia52B.E.coliMedia55D.SolutionFormulations56E.PlasmidInformation60F.YeastHostStrainInformation63TableofContentsYeastProtocolsHandbookProtocolNo.PT3024-1β-galactosidaseAssays25TableIV.SelectedYeastGenesandTheirAssociatedPhenotypes51TableV.MatchmakerReporterGenesandTheirPhenotypes51TableVI.MatchmakerTwo-HybridSystemCloningVectors60TableVII.MatchmakerTwo-HybridSystemReporterandControlPlasmids61TableVIII.MatchmakerOne-HybridSystemCloning,Reporter&ControlPlasmids62TableIX.YeastReporterStrainsintheMatchmakerOne-andTwo-HybridSystems63ListofFiguresFigure1.SequenceofGAL4DNA-BDrecognitionsitesintheGAL1,GAL2,MEL1UASsandtheUASG17-mer6Figure2.Urea/SDSproteinextractionmethod14Figure3.TCAproteinextractionmethod16TableofContentscontinuedNoticetoPurchaserClontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,invitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.NOTICETOPURCHASER:LIMITEDLICENSEUseofthisproductiscovered6,127,155.Thepurchaseofthisproductincludesalimited,non-transferableimmunityfromsuitundertheforegoingpatentclaimsforusingonlythisamountofproductforthepurchaser’sowninternalresearch.Norightunderanyotherpatentclaim(suchasmethodclaimsinU.S.PatentsNos.5,994,056and6,171,785)andnorighttoperformcommercialservicesofanykind,includingwithoutlimitationreportingtheresultsofpurchaser’sactivitiesforafeeorothercommercialconsideration,isherebyconveyedbythepurchaseofthisproductexpressly,byimplication,orbyestoppel.Thisproductisforresearchuseonly.DiagnosticusesrequireaseparatelicensefromRoche.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.Clontech,theClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.,unlessnotedotherwise.ClontechisaTakaraBioCompany.©2009ClontechLaboratories,Inc.ContactUsForAssistanceCustomerService/Ordering:TechnicalSupport:Telephone:[phone](toll-free)Telephone:[phone](toll-free)Fax:[phone](toll-free)Fax:[phone]Web::[e-mail]-mail:[e-mail]kClontechLaboratories,Inc.™Two-HybridandOne-HybridSystems,andMatchmakerLibraries.TheYeastProtocolsHandbookisespeciallyusefulforresearcherswhowishtouseyeastasavehiclefortheirmolecularbiologyexperiments,buthavelittleornopriorexperienceworkingwithyeast.Fornoviceandexperiencedusersalike,theYeastProtocolsHandbookwillhelpyouobtainthebestpossibleresultswithyourMatchmakerandotheryeast-relatedproductsfromClon