Curing strains of endogenous 2micron plasmid
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!
免责声明 本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘”。违反上述声明者,本网将追究其相关法律责任。