LindaHoskins/HahnLab

LastmodifiedWed,Jul5,2000

Thismoreinvolvedmethodgiveshighertransformationefficiencythanthesemirapidmethod.UseDMSOiftransformingalibrarytoyeastand/ortransformingamutantstrainwhichtransformspoorlyusingtheLiOAcmethod.

1.Inoculate5mlsYPDwithasingleyeastcolony.GrowO/Nat30degreesC.2.Add0.5mlscultureto4.5mlsfreshmedia,checkA660.Addsuitableamountofcellsto60mlsfreshmediatogiveA660=0.2(2x106cells/ml).GrowtoA660=1.0(2x107cells/ml),takesapproximately5hours.3.Spindown50mlscells,washin10mlssterilewater,recentrifuge,resUSPendin1mlsterilewater.Transferto1.5mlsterilemicrofugetube,spindown,resuspendin1mlsterileTE/LiOAc(madefreshfrom10XTE[0.1MTris-HCl,0.01MEDTA,pH7.5]and10XLiOAc[1MLiOAcpH7.5,adjustedwithdilutedaceticacid]).Spindown,resuspendin0.25mlsTE/LiOAc(4x10⁹cells/ml).4.Mix50ulyeastcellswithtransformingDNAand5ulsinglestrandedcarrierDNA(10mg/ml,boiledandquickchilledonice)ina1.5mlmicrofugetube.5.Add300ulsterilePEG(40%PEG4000,1XTE,1XLiOAc,madefreshfromsterile50%PEG4000,10XTE,and10XLiOAc).Mixthoroughly.6.Incubateat30degreesCfor60minuteswithoccasionalgentleshaking.7.Add40ulDMSO,mixthoroughly.(Thisincreasestransformation5-10fold.)

8.Heatshockat42degreesCfor15minutes.

9.Microfuge10seconds,removesupernatant.Resuspendin1ml1XTE.Microfuge10sec.Resuspendin1ml1XTE.Plate200ulonselectivemedia.