Procedure

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 1-5 minutes.

2. Add 200 µl of Harju- buffer

3. Immerse tubes in a dry ice-ethanol bath for 2 minutes,

4. Transfer to in a 95°C water bath for 1 minute.

5. Repeat the last two steps

6. Vortex 30 seconds.

7. Add 200 µl of chloroform and vortex 2 minutes.

8. Centrifuge 3 minutes at room temperature, 20,000 × g.

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

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

11. Centrifuge 5 minutes at room temperature, 20,000 × g.

12. Remove the supernatant with a pulled Pasteur pipette by vacuum aspiration.

13. Wash the pellet with 0.5 ml 70% ethanol

14. Centrifuge 5 minutes at room temperature, 20,000 × g.

15. Remove supernatant.

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

17. 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