ThisProtocolallowsYoutoprepareFrozenYeastcellsthatarecompetentfortransformationafterthawing

AfterDohmenetal.(1991)Yeast7:691-692.SeeSchiestletal(1993)

TheABIlitytomakeyeastcellscompetentfortransformationinadvanceallowsthoselaboratoriesworkingwithfewstrainstopreparelargebatchesofcellsforlatertransformation.Thisisconvenientforlaboratoriesusingthetwo-hybridandothersimilarsystems.Yeastcellscanbemadecompetentfortransformationbytreatmentwithethyleneglycolanddimethylsulfoxide(DMSO)andthenfrozeninsmallaliquotsandstoredat-70^oC.Whilethehighesttransformationefficienciesareobtainedwithfreshlygrowncultures,themoderatelyefficienttransformationoffrozencompetentcellssavestime.Thisprocedurehasbeenreportedtoproduceupto105transformants/µgplasmidDNA(Dohmenetal.1991).

1. GrowcellsinYPAD(10mlpertransformation)toanOD₆₀₀or0.6to1.0.Thisrepresentsacelldensityofapproximately0.6-1x10⁷cell/ml.
2. Washthecellsin0.5volof1.0M.sorbitol,10mMBicine-NaOH(pH8.35),3%ethyleneglycol,5%DMSO(Solution1),andresUSPendin0.02volofthesamesolution.
3. Freezethe0.2mlaliquotsslowlyusingaNalgeneCryo1^oCfreezingcontainer(Cat.No.5100-0001)andstoreat-70^oCuntilneeded.

   TIP:

   Slowfreezingresultsingoodviability.

   • Add0.1-5µgofplasmidDNAand50µgofsinglestrandedcarrierDNA(10mg/ml)inamaximumvolumeof20µlontopofthefrozencellsuspension.
   • Placeina37^oCwaterbathandmixevery10-15secuntilthesolutionbeginstomelt.Removefromwaterbathandmixuntilmeltingiscomplete.
   • Add1.4mlofSolution2(40%PEG1000(Roth,Karlsuhe,Germany),0.2MBicine-NaOH(pH8.35))andmixbyvortexingfor1min.
   • Incubateat30^oCfor1hr.
   • Spindownthecellsat3000xgfor5secandresuspendthecellpelletin1.0mlofSolution3(0.15MNaCl,10mMBicine-NaOH(pH8.35)).
   • PlateanappropriateamountontoSCommissionmediumintoapuddleofSolution3.

   Preparationoffrozencompetentcellsfromthetwo-hybridstrainPJ69-4Ausingthemethodaboveroutinelygaveresultsbetween0.9-1.0x10⁴transformants/µg.