3-36-ppic9k-操作手册

UserManualCorporateHeadquartersInvitrogenCorporation1600FaradayAvenueCarlsbad,CA92008T:17606037200F:17606026500E:[e-mail]-specificcontactinformationvisitourwebsiteat–20VersionG03June201025-0106iiTableofContentsKitContentsandStorage.............................................................................................................................vAccessoryProducts....................................................................................................................................viIntroduction.......................................................................................................................................................1Overview.......................................................................................................................................................1Materials........................................................................................................................................................5Methods.......................................................................................................................................................6CloningintopPIC9K....................................................................................................................................6AnalysisofE.coliTransformants...............................................................................................................9TransformationintoPichia........................................................................................................................10InVivoScreeningofMultipleInserts.......................................................................................................12AnalyzingResults......................................................................................................................................16Troubleshooting..........................................................................................................................................17Appendix.....................................................................................................................................................18Recipes.........................................................................................................................................................18PichiaGenomicDNAIsolation.................................................................................................................19Easy-DNA™ProtocolforIsolationofDNAfromPichia........................................................................21DeterminationofCopyNumberofMultipleIntegrants......................................................................22MapandFeaturesofpPIC9K...................................................................................................................24TechnicalSupport.......................................................................................................................................26PurchaserNotification...............................................................................................................................27References....................................................................................................................................................28iiiivKitContentsandStorageShippingandStoragepPIC9Kisshippedonwetice.Uponreceipt,storepPIC9Kat–20°C.ContentsThiskitcontains:20μgofpPIC9Kvector,suppliedinsuspensionas40μlof0.5μg/μlvectorin10mMTris–HCl,1mMEDTA,pH8.0vviAccessoryProductsAdditionalProductsTheproductslistedinthissectionareintendedforusewiththepPIC9Kvectors.Formoreinformation,visitourwebsiteat(page26).ProductQuantityCatalogno.OriginalPichiaExpressionKit1kitK1710–01EasySelect™PichiaExpressionKit1kitK1740–01Easy-DNA™Kit15–200reactionsK1800–01TACloning®Kit20reactionsK2000–01pPIC3.5K20μgeachV173–20pAO81520μgeachV180–20PichiaEasyComp™TransformationKit1kitK1730–01PichiaProtocols1bookG100–01OneShot®TOP10(chemicallycompetentE.coli)10reactions20reactionsC4040–10C4040–03OneShot®TOP10ElectrocompetentE.Coli10reactions20reactionsC4040–50C4040–52TOP10Electrocomp™Kits20reactions2×20reactionsC664–55C664–11AmpicillinSodiumSalt,irradiated200mg11596–027Geneticin®SelectiveAntibiotic(powder)5g25g11811–03111811–098Geneticin®SelectiveAntibiotic(liquid)20ml100ml10131–03510131–027IntroductionOverviewIntroductionMultiplecopyintegrationofrecombinantgenesinPichiahasbeendemonstratedtoincreaseexpressionofthedesiredgeneinsomecases(Brierley,etal.,1994;Clare,etal.,1991a;Cregg,etal.,1993;Romanos,etal.,1991;Scorer,etal.,1993;Scorer,etal.,1994;Thill,etal.,1990;Vedvick,etal.,1991.).ThepPIC9Kvectorincludedinthiskitallowsisolationofmulticopyinsertsbyaninvivomethod,inordertotestwhetherincreasingthecopynumberofyourrecombinantgenewillleadtoasubsequentincrease