Yeast DNA Extraction for Southern Blot
References: Hoffman & Winston (1987). Gene 57: 267 – 272.
Jenn Gallagher
General Precautions:
 Phenol should be used in the hood with gloves.
 Try not to excessively vortex the DNA (with the exception of the vortex step). It will
cause excessive shearing.
DNA Isolation:
 Grow 10ml yeast prep overnight to saturation in YPD. Add Ade if strains are ade
Spin cells 5 min, 3000 rpm

Resuspend cells in 500 µl H2O. Transfer to a microfuge tube and spin down 5 sec.

Decant gently and vortex tube briefly to resuspend cells in residual liquid.

Add 200 µl Lysis Buffer
o
Add 200 µl Phenol/CHCl3/IAA
o
Add 0.3 g acid washed beads (0.45 – 0.52 mm)

Vortex for 3 – 4 min. Do not bead beat. Add 200 µl TE. Mix.

Spin 5 min at top speed in microfuge. Transfer aqueous layer to a fresh tube

Add 1ml 95% Ethanol. Invert to mix. (Optional: Freeze @ -20C for 15 min).

Spin down DNA 2 min in microfuge. Resuspend pellet in 400 µl TE + 3 µl 10 mg/ml
RNaseA. 5 min at 37C.

Add 40 µl 3 M NaOAc pH 5.7 and 1 ml 95% Ethanol. Invert to mix (Optional
Freeze @ -20C for 15 min).

Spin 2 min in a microfuge. Wash with 70% Ethanol. Dry.

Resuspend in 20 – 50 µl TE.
Lysis Buffer
Reagent
10 % SDS
5 M NaCL
1 M Tris pH=8.0
0.5 M EDTA
Triton X-100
Total Volume
Final Conc.
1%
100 mM
10 mM
1 mM
2%
Amount
5 ml
1 ml
0.5 ml
100 µl
1 ml
50 ml
11/30/07
Erin Osborne