(seeClontechYPH,p31) Pickcoloniesinto0.5mlofSD-Leu(orotherappropriateSDmedium) Vortexfor1min LeavetogrowO/Nfor18-24hat30°C,230-250rpm(bestin5mlbijou) Spinyeastcultureat13,000rpmfor5min(microfuge) PouroffsupernatantcarefullyandresUSPendthepelletinresidualliquid(~50µl) Add10µlLyticase(SigmaL2524at5Units/µlinTE;aliquotsstoredinat-20°C) Mixthoroughlybyvortexing/pipetting Incubate1hat37°C,250rpm Add10µlof20%SDSandvortexfor1min Freezesamplesat-20°C Thaw Vortex StartQiagenminiprepprotocolbyadding180µlofBufferP1toobtainafinalvolumeof250µl Add250µlBufferP2 Etc..followQIAGENprotocol EluteDNAin30µlofH2O Use20µlofelutedDNAtotransform200µlcompetentXL1-Blues PlateonLB/Amp,growupfromcoloniesandminiprep. Alternatively:Inoculatedirectto5mlLB/AmpO/NandontoLB/Ampplates50:50.IsolateplasmidusingQIAGENminipreps.Plasmidisolationfromyeast
