抗生素英文课件精品cultivation and transformation of

TianHe CultivationandTransformationofYeasts Media non selective YPDwithoutantibioticsSelective Syntheticcompletedropoutmedium SC auxotroph YPDwithantibiotics YPD 1000mLYeastExtract10gPeptone Tryptone 20gGlucose20gAgar forplates 20gDistilledH2O1000mL Syntheticcompletedropoutmedium SC 1000mLYeastnitrogenbasewithoutaminoacids YNB 6 7gDropoutmix His Trp LeuDOmix 0 62gAminoacidsGlucose2gAgar forplates 2gDistilledH2O1000mL Cultivation Temperature 30 Lower yeastsgrowslowly Higher yeastsdie Shake 200rpmorhigherAvoidthecontaminationofE Coli Glovesareneededtoavoidinfection Notes Ittakes2hoursormoretocompleteacycle Yeastwillage Inoculateanewplateeverymonth Theselectablemarkerandmediumshouldbecomplementary CommonlyusedselectablemarkerLEU2 URA3 TRP1 HIS3 ADE1 2pGREG503504505506HISTRPLEUADE Question whyweusepGREG503forhomologousrecombination Wecannotdeterminefromthephenotypewhetherrecombinationoccurs pGREG503 4 5 Autonomouslyreplicatingsequence Auxotrophmarker Antibioticmarker Antibioticmarker Homologoussequence Stuffer Promoter Tag Yeaststrain AH109 GAL4inducedpromoters GAL1 GAL2 MEL1 Transformation Plasmid ssDNA dsDNAThecompetentcellsofyeastcannotbestored itmustbepreparedbeforeuse Materials TE 0 1MTris HCl 0 01MEDTA pH7 5LiAc 1MLiAc pH7 5PEG MW4000 50 w v storedatroomtemperature Cappedsecurelytoavoidevaporation Single strandedCarrierDNA 2 0mg mL SalmonspermDNA Boil1 0mLcarrierDNAfor5minutesandquicklychillonicewater DonotboilthecarrierDNAeverytime Keepasmallaliquotinfreezerboxandboilafter3 4freezethaws Keeponicewhenout Protocol Inoculatecellsinto50mLYPDandgrowovernighttoadensityof1 2 107 mL nearlysaturated Asuspensioncontaining1 106cells mLgivesanOD600of0 1 Diluteto2 106 mLinfreshYPDandre growintoexponentialphase 1 107 mL Ittypicallytakes3 4hours Itisimportanttoallowthecellstocompleteatleast2divisions Transformationefficiencyremainsconstantfor3 4cycles Harvestthecultureinasterilecentrifugetubeat2500rpmfor5minutes Washinsterilewatertwice Resuspendin1 0mLsterilewaterandtransferto1 5mLmicrofugetube centrifugefor30sat13 000ganddiscardthesupernatant Protocol Washcellsin1 0mLofTE LiAc 10 andresuspendat2 109cells mLinTE LiAc 1 Mix50 L 1 108cells with1 gtransformingDNAand50 gsingle strandedcarrierDNAinmicrofugetubes Add300 Lsterileplatesolution 40 PEG4000 1 TE LiAc for1mLplatesolution youshouldadd0 8mL50 PEG4000 0 1mL10 TE 0 1mL10 LiAc Vortextomixthoroughly Incubateat30 intheshakerfor30minutes Heatshockina42waterbathfor15minutes differentstrainshavedifferentoptimalheatshocktime Protocol Centrifugeat13 000gfor30s Removethesupernatantcarefully