Curingstrainsofendogenous2-micronplasmid

Inordertocreateacircle-zeroderivativefromaCir⁺strain,themethodofRoseandBroach(1990)isused.A2-micron-basedplasmid,YEp351-GAL-FLP1*,thatcarriestheLEU2selectableMarkerandalsotheFLP1geneunderthecontroloftheGALpromoteristransformedintoyeast.Leu⁺transformantsaregrownonmediumcontaininggalactoseasthecarbonsource.Undertheseconditions,theFLP1geneisoverexpressedandcausesover-replicationoftheendogenousandartificial2-micronplasmids,whichisdeleterioustocells.Therefore,cellsthathavelosttheplasmidshaveasignificantgrowthadvantageoverthosethatretainthem.

1. TransformyeastwithYEp351-GAL-FLP1,andselecttransformantsonSC-leu-glu2%GAL(galactose)medium.
2. PatchtransformantsontoSC-leu-glu2%GALmedium.
3. Patchesthatgrowwellarecandidatesforstrainsthathavelostthe2-micronplasmid.Growculturesfromthesepatchesovernightin5mlofYPD.
4. Platecellsfromthegrownculturesatadensityof~200coloniesperYPDplateandallowthemtogrowintocolonies.
5. ReplicaplatetoSC-leutoidentifythoselackingYEp351-GAL-FLP1.(CellsthathavelostYEp351-GAL-FLP1shouldnotbeabletogrow).
6. 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!

================  蚂蚁淘在线  ================

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：未经蚂蚁淘在线授权不得转载、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：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。