DNA Preparation from 10-mL Yeast Culture

Contributor: Namjin Chung
Date: April 20, 1996

1. Grow 10-mL yeast cultures overnight to saturation in appropriate media at 30 C.

2. Spin cells down for 5 min at 1,500 rpm (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 200 uL Yeast Lysis Buffer (see below for recipe), 200 uL
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 200 uL TE (pH 8).

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

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

9. Add 10 uL 4M ammonium acetate and 1 mL 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 50 uL TE.

Yeast Lysis Buffer (Winston Lysis Buffer) 200 mL
Triton X-100 4 mL
10% SDS 20 mL
5M NaCl 4 mL
0.5M EDTA 400 uL
1M Tris (pH 8) 2 mL
distilled water to 200 mL

Reference
Hoffman and Winston (1987) Gene 57: 267-272.