Springer Lab: GenomicDNAIsolation

Procedure

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.Immerse tubes in a dry ice-ethanol bath for 2 minutes,

4.Transfer to in a 95°C water bath for 1 minute.

5.Repeat the last two steps

6.Vortex 30 seconds.

7.Add 200 µl of chloroform and vortex 2 minutes.

8.Centrifuge 3 minutes at room temperature, 20,000 × g.

9.Transfer the upper aqueous phase to a microcentrifuge tube containing 400 µl ice-cold 100% ethanol. Mix by inversion or gentle vortexing.

10.Incubate at room temperature, 5 minutes. Alternatively, precipitate DNA at -20°C to increase yield.

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

12.Remove the supernatant with a pulled Pasteur pipette by vacuum aspiration.

13.Wash the pellet with 0.5 ml 70% ethanol

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

15.Remove supernatant.

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

17.Resuspend in 25- 50 µl TE (pH 8.0)] or water. Samples obtained directly from plates should be resuspended in a 10 µl volume, because the yield will be smaller. 0.25 µl RNase cocktail should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).

18. (Optional) Add ~1 microL of RNAase A to each sample

Reagents

Harju- Buffer – 2% Triton X-100 – 1% SDS, – 100 mM NaCl – 10 mM Tris-HCl, pH 8.0, – 1 mM EDTA

Reference

Harju S, Fedosyuk H, Peterson KR. Rapid isolation of yeast genomic DNA: Bust n' Grab. BMC Biotechnol. 2004 Apr 21;4:8.; PMID: 15102338