SMALLScaleTransformation

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.

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