Procedure

Transfer 1.5 ml of liquid culture of yeast grown for 20 - 24 h at 30°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) into a microcentrifuge tube. Pellet cells by centrifugation at 20,000 × g for 1-5 minutes. Add 200 µl of Harju- buffer Immerse tubes in a dry ice-ethanol bath for 2 minutes, Transfer to in a 95°C water bath for 1 minute. Repeat the last two steps Vortex 30 seconds. Add 200 µl of chloroform and vortex 2 minutes. Centrifuge 3 minutes at room temperature, 20,000 × g. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 µl ice-cold 100% ethanol. Mix by inversion or gentle vortexing. Incubate at room temperature, 5 minutes. Alternatively, precipitate DNA at -20°C to increase yield. Centrifuge 5 minutes at room temperature, 20,000 × g. Remove the supernatant with a pulled Pasteur pipette by vacuum aspiration. Wash the pellet with 0.5 ml 70% ethanol Centrifuge 5 minutes at room temperature, 20,000 × g. Remove supernatant. Air-dry the pellets at room temperature or for 5 minutes at 60°C in a vacuum dryer. Resuspend in 25- 50 µl TE (pH 8.0)] or water. Samples obtained directly from plates should be resuspended in a 10 µl volume, because the yield will be smaller. 0.25 µl RNase cocktail should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).

Reagents

Harju- Buffer � 2% Triton X-100 � 1% SDS, � 100 mM NaCl � 10 mM Tris-HCl, pH 8.0, � 1 mM EDTA