酵母DNA的提取

1. Grow 10-ml yeast cultures overnight to saturation in appropriate media at 30℃.

2. Spin cells down for 5min at 1,500rpm (Beckmann), and resuspend the cells in 0.5 ml sterile distilled water.

3. Transfer the cells to 1.5-ml tube, and spin them down for 5 sec in the Eppendorf centrifuge at the maximum speed (14,000 rpm).

4. Decant the supernatant and briefly vortex the pellet in the residual water.

5. In the following order, add 200μL Yeast Lysis Buffer (see below for recipe), 200μL phenol:chloroform:isoamyl alcohol (25:24:1), and 0.3 g of acid-washed glass beads (Sigma ).

6. Vortex for 3-4 min, and add 200μl TE (pH 8).

7. Spin for 5 min in a microfuge, and transfer aqueous phase to a new tube. Add 1mL 100% EtOH, and mix by inversion.

8. Spinf for 1 min in a microfuge, and aspirate the supernatant. Resuspend the pellet in 400μl TE and 4μL RNase A (Boerhinger Mannheim ), and incubate until the pellet is dissolved at 37℃.

9. Add 10μl 4M ammonium acetate and 1mL 100% EtOH.

10. Spin for 1 min in a microfuge, and discard the supernatant. Wash with 1ml 70% EtOH.

Air-dry the pellet and resuspend in 50μl TE.

Yeast Lysis Buffer (Winston Lysis Buffer) 200ml

Triton X-100 4ml

10% SDS 20ml

5M NaCl 4ml

0.5M EDTA 400μl

1M Tris (pH 8) 2ml

distilled water to 200ml