Team:UNICAMP-Brazil/Protocols/Yeast DNA Extraction

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Yeast DNA extraction

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. Add ~300 µl glass beads. Add 200 µl of phenol /chloroform/ IAA (25:24:1).

4. Vortex 2 minutes.

5. Centrifuge 3 minutes at 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 on ice temperature, 1 hour.

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

9. Remove the supernatant

10. Wash the pellet with 0.5 ml 70% ethanol

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

12. Remove supernatant.

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

14. Resuspend in 25- 50 µl water.

Harju- Buffer

– 2% Triton X-100

– 1% SDS,

– 100 mM NaCl

– 10 mM Tris-HCl, pH 8.0,

– 1 mM EDTA