Yeast Genomic DNA “Miniprep”

1. Grow a 3-15 ml overnight to stationary phase or desired OD₆₀₀.

2. Spin, remove supernatant.

3. Wash cells with H₂O.

4. Resuspend washed cells in 100 ml DNA PREP buffer. Add Zymolyase to 1 mg/ml,

b-mercaptoethanol to 1%. It’s convenient to make a mix of the above ingredients when you have several samples.

5. Incubate at 37°C for 30’-40’. Occasionally shake.

6. Add 400 ml LYSIS buffer and mix gently by inversion.

7. Incubate at 70°C for 20’.

8. Transfer the tube to ice; add 100 ml 5M KOAc and mix by inversion.

9. Incubate on ice for at least 30’.

10. Spin in mfuge for 5’ to pellet cellular debris.

(optional: RNase treat)

11. Pour the supernatent into a mfuge tube containing 200 ml phenol:chloroform (1:1). Mix by vortexing at medium speed, approximately 5 seconds.

12. Spin 3-5’ in a mfuge to serparate phases. Transfer 550 ml of the aqeous phase to a fresh tube containing another 200 ml phenol:chloroform (1:1). Extract as above.

13. Remove 500 ml of the aqeous phase to a new mfuge tube containing 1 ml 95% ethanol, mix.

14. Precipitate the DNA by incubating the sample at -20°C for 10’.

15. Pellet, rinse with 70% ethanol and dry the DNA.

16. Resuspend the DNA in 50 ml TE. Hint: If it gets too dry, the DNA pellet is difficult to resuspend)

DNA PREP Buffer LYSIS Buffer

1M sorbitol 0.1M Tris base (pH 9.7)

0.1M sodium citrate, pH 7.0 0.5% SDS

60mM EDTA 50mM EDTA

From Nasmyth and Reed (1980). PNAS USA, 77, 2119-2123.