酵母单杂交系统及其实验操作.ppt

《酵母单杂交系统及其实验操作.ppt》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。

1、YeastOneHybridSystemI.IntroductionII.YeastMolecularTechniquesIII.ConstructingaReporterVectorforOne-HybridAnalysisIV.GeneratingacDNALibraryV.Constructing&ScreeningaOne-HybridLibraryVI.AnalyzingPositiveInteractionsYeastonehybridsystemI.IntroductionOne-HybridSystemprovides

2、aninvivogeneticassayusedforisolatingnovelgenesencodingproteinsthatbindtoatarget,cis-actingregulatoryelementProtein-DNAinteractionI.INTRODUCTIONThedifferencesbetweenyeastone&twohybridII.YeastMolecularTechniquesYeaststrainsA.GenotypesB.PhenotypesII.YeastMolecularTechnique

3、sYeaststrain：Y187（MATα）Can’tsynthesisHis、Leu、Trpbyitself。YeastIncubateYeastselectionmedium-Hisor-Leuor-TrpYeastcan’tgrowthonselectionmediumII.YeastMolecularTechniquesYeaststrainsC.MatingtypecompatibilitiesY187(MATα)canmatewithAH109,HF7c,CG-1945,Y190,orSFY526(allMATa).D.

4、ReportergenesGAL1UASGALTATALacZII.YeastMolecularTechniquesYeaststrainsE.LeakyHIS3expression3-AT:3-amino-1,2,4-triazoleisacompetitiveinhibitoroftheyeastHIS3protein(His3p).3-ATisusedtoinhibitlowlevelsofHis3pexpressedinaleakymannerandthustosuppressbackgroundgrowthonSDmediu

5、mlackinghistidine.SomeyeaststrainshaverelativelyhighbasallevelsofHis3p.II.YeastMolecularTechniquesMaindifferencebetweenyeastandE.colicultureYeastE.Coli4~6days16hoursYeastE.Coli30℃37℃YeastE.ColiYPDLBIncubationtimeIncubationtemperatureCulturemediumTooptimizethe3-ATconcent

6、rationinyourselectionmedium:Usethelowestconcentrationof3-ATthat,afteroneweek,allowsonlysmall(<1mm)coloniestogrow.Toomuch3-ATinthemediumcankillfreshlytransformedcells.YEASTMEDIAA.YPD(YPDA)medium20g/LDifcopeptone10g/LYeastextract20g/LAgar(forplatesonly)(To0.003%0.2%adenin

7、ehemisulfateSolution)pHto6.5To2%glucosesterile(40%stocksolution)II.YeastMolecularTechniquesYEASTMEDIAB.SDmedium(1L)6.7gYeastnitrogenbasewithoutaminoacids20gAgar(forplatesonly)850mlH2OpHto5.8100mloftheappropriatesterile10XDropoutSolution3-AT(1M3-ATstocksolution)To2%gluco

8、sesterile(40%stocksolution)II.YeastMolecularTechniquesC.10XDropout(DO)SolutionNutrient10XConcentrationL-Adenin