Table 1 Bust n' Grab Protocol

1. 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 5 minutes.

2. Add 200 μl of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0).

3. Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to in a 95°C water bath for 1 minute. Repeat; vortex 30 seconds.

4. Add 200 μl of chloroform; vortex 2 minutes.

5. Centrifuge 3 minutes, room temperature, 20,000 × g.

6. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 μl ice-cold 100% ethanol. Mix by inversion or gentle vortexing.

7. Incubate at room temperature, 5 minutes. Alternatively, precipitate at -20°C to increase yield.

8. Centrifuge 5 minutes, room temperature, 20,000 × g. Remove supernatant with a pulled Pasteur pipette by vacuum aspiration.

9. Wash the pellet with 0.5 ml 70% ethanol, spin down as described in step 8 above. Remove supernatant.

10. Air-dry the pellets at room temperature or for 5 minutes at 60°C in a vacuum dryer.

11. Resuspend in 25–50 μl TE [10 mM Tris (pH 8.0), 1 mM EDTA (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 (Ambion) should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).