Curing of Saccharomyces cerevisiae 2μm DNA by...
来自 : 蚂蚁淘
Curingstrainsofendogenous2-micronplasmid
Inordertocreateacircle-zeroderivativefromaCir+strain,themethodofRoseandBroach(1990)isused.A2-micron-basedplasmid,YEp351-GAL-FLP1*,thatcarriestheLEU2selectableMarkerandalsotheFLP1geneunderthecontroloftheGALpromoteristransformedintoyeast.Leu+transformantsaregrownonmediumcontaininggalactoseasthecarbonsource.Undertheseconditions,theFLP1geneisoverexpressedandcausesover-replicationoftheendogenousandartificial2-micronplasmids,whichisdeleterioustocells.Therefore,cellsthathavelosttheplasmidshaveasignificantgrowthadvantageoverthosethatretainthem.
- TransformyeastwithYEp351-GAL-FLP1,andselecttransformantsonSC-leu-glu2%GAL(galactose)medium.
- PatchtransformantsontoSC-leu-glu2%GALmedium.
- Patchesthatgrowwellarecandidatesforstrainsthathavelostthe2-micronplasmid.Growculturesfromthesepatchesovernightin5mlofYPD.
- Platecellsfromthegrownculturesatadensityof~200coloniesperYPDplateandallowthemtogrowintocolonies.
- ReplicaplatetoSC-leutoidentifythoselackingYEp351-GAL-FLP1.(CellsthathavelostYEp351-GAL-FLP1shouldnotbeabletogrow).
- Toverifythelossofthe2-micronplasmids,preparegenomicDNAfromLeu-strains(asdescribedinSectionVII)andperformSouthernanalysis(SectionVIII)onundigestedDNAusingDNAofYEp351-GAL-FLP1asaprobe.IncludeDNAfromaCir+strainonthesamegelasapositivecontrol.
PeoplealwaysaskusforamapofYEp351-GAL-FLP1,butwedon"thaveone.Allweknowis,it"sYEp351based.Linearizeitwithsomethingandlabelthewholething.
Errors!
- Inpreviouspublications,wehaveerroneouslydesignatedtheYEp351-GAL-FLP1plasmidaspFV17.pFV17isanintegratingplasmiddescribedinthesameRoseandBroachpaper.
- Inpreviousversionsofthiswebpage,wesaidtheplasmidcontainedGAL-REP1.Whoops!
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