1.Inoculate1mlofYPDAorSDwithseveral2–3mmcolonies. 2.Vortexvigorouslytodisperseanyclumps. 3.Transfercellstoaflaskcontaining50mlofYPDAorSD: 4.Incubateat30oCfor16–18hrwithshaking(250rpm)tostationaryphase(OD600>1.5). 5.Transferovernightculture(enoughtoproduceanOD600=0.2–0.3)into300mlofYPDA 6.Incubateat30oCfor3hrwithshaking(230–270rpm). 7.Placecellsin50-mltubesandcentrifugeat1,000xgfor5minatroomtemperature. 8.DiscardthesupernatantandresUSPendcellpelletsbyvortexingin25–50mlofsterileTEorH2O 9.Poolcellscentrifugeat1,000xgfor5minatroomtemperature. 10.Decantthesupernatant. 11.Resuspendthecellpelletin1.5mloffreshlyprepared,sterile1XTE/LiAc: 12.Prepare10mlPEG/LiAcsolution. 13.Intheindicatedtube,mixthefollowinga: •DNA-BD/bait0.1µg •AD/library0.1µg •HerringtestescarrierDNA0.1mg(10µl) 14.Add0.1mlofyeastcompetentcellsandmixwellbyvortexing. 15.Add0.6mlofsterilePEG/LiAcsolutionandvortexathighspeedtomix. 16.Incubateat30oCfor30minwithshaking(200rpm). 17.Add70µlofDMSO.Mixwellbygentleinversionorswirling.Donotvortex. 18.Heatshockfor15minina42oCwaterbath. 19.Chillcellsonicefor1–2min. 20.Centrifugecellsfor5secatroomtemperatureat14Krpm 21.Removethesupernatant. 22.Resuspendcellsin0.5mlof1XTEb: 23.Proceedtoplating(oneitherSD–Trp/–Leu)orSD–Trp/–Leu/–His/–AdewithorwithoutX-á-galasrequired.SMALLScaleTransformation