(seeClontechYPH,p12) Crackingbufferstocksolution(seep54ClontechYPH) 8MUrea;5%w/vSDS;40mMTris-HClpH6.8;0.1mMEDTA;0.4mg/mlBMBinH2O forafinalvolumeof20ml9.6gUrea 1gSDS 0.8ml1MTrisHClpH6.8 4µl0.5MEDTA 8mgBromophenolBlue Protocol 1.Setup5mlO/NculturesincorrectSDselectionmedium(SD-LeuorSD-Trp). 2.Alsosetupuntransformedyeastasacontrol.Leaveat30°C,230-250rpm,for18-24h. 3.Foreachclonetobeassayed,separatelyinoculate50mlofYPDAwiththeentireO/Nculture.TakeanOD600reADIng. 4.Incubateat30°C,230-250rpm,untiltheOD600reaches0.4-0.6(thiscantakebetween4-8h). 5.UseOD600Unitstodeterminethevolumeofeachyeastculturetobetaken;i.e.decidetotake15OD600Unitsofeachculture15/OD600=volumetobetaken,soifOD600=0.5,take30ml 6.Foreachculture,transferthevolumecalculatedaboveintoa50mlFalconhalffilledwithice.Spinat2500rpm(MSE)for5min. 7.PouroffsupernatantandresUSPendpelletinto50mloficecoldH2Oandspinagain. 8.RemovesupernatantandfreezethepelletinliquidN2(cellscanbestoredat-80°C) 9.Justbeforeuse,makeupcompletecrackingbufferPreparationofYeastProteinExtracts
