-Usesteriletechniqueandsterilesolutionsthroughoutthismethod.-1.Ina15mlculturetube,inoculate2-3mloftheappropriatemediawithasinglecolonyoftheyeaststraintobetransformed.Thisisthestarterculture.***Variouswild-typeandmutantyeaststrainscanbeobtainedfromResearchGenetics(Huntsville,AL)orATCC(Manassas,VA).2.Growthestartercultureat30withshaking(250rpm)untilitreachessaturation.***Thistakesanywherefrom1to6days,dependingonthestrainandthemedia.3.Place0.5mlofthesaturatedcultureinasterilemicrofugetube.4.Collectthecellsbycentrifugingat16,000xgfor30sec.5.Aspiratethesupernatant.6.Add10mlofsonicatedsalmonspermDNA(10mg/mlstock)(Stratagene,#201190).***TheDNAmustbesingle-stranded,whichcanbeachievedbyboilingfor5min(a100°Cheatblockworkswell)andimmediatelychillingonice.***TheDNAonlyneedstobeboiledevery3to4timesitisused(aslongasitremainsonicewhenthawed).7.Add1-2mgoftheplasmidDNAtobetransformedandvortex.8.Add500mlofPLATEsolution.Mixbyinvertingorbypipettinggently.Donotvortex.PLATEsolution40%PEG3350(w/v)100mMlithiumacetate(LiAc)10mMTris,pH7.50.4mMEDTA9.Leaveatroomtemperatureorat30for24-48hrs.10.Collectthecellsbycentrifugingat16,000xgfor30sec.11.Aspiratethesupernatant.12.ResUSPendthecellsin200mlofsterilewaterbypipettinggentlyandthoroughly.13.Spreadonsolidmediathatwillselectfortheplasmid.14.Incubateat30°untilcoloniesappear.***Thistakesaround2to6days,dependingontheyeaststrainandtheplasmid"snutritionalMarker.Inourexperience,itusuallytakes~2daysforLEU2plasmidsand~6daysforURA3plasmids.15.Restreak2-3transformedcoloniesontoanewplate.

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