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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
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