Fast small scale isolation of yeast total DNA (Method of Wach et al.)

Wach, A., Brachat, A., Pöhlman, R. and Philippsen, P. (1994). New heteroloogous models for classical or PCR-based gene disruptions in Saccharomyces serevisiae. Yeast, 13 : 1793-1808

Day 1

1. Grow a yeast culture overnight in 2 ml YPD at 30°C to early stationary growth phase (» 2 x 10⁸ cells/ml) or grow a yeast culture overnight in 8 ml minimal medium (supplemented with 50 mM sodium phosphate pH 6,4) to a density of » 5 x 10⁷ cells/ml (OD₆₀₀ » 1)

Day 2

1. Collect cells from 1.3 ml of the YPD culture in a microcentrifuge tube by centrifugation (6000 rpm for 3 min)
   or
   Harvest cells from the total minimal medium culture volume (at a density of » 5 x 10⁷ cells/ml)
2. Resuspend the cells in 1 ml of sterile water
   Harvest cells by centrifugation (6000 rpm for 3 min)
3. Resuspend the cells in 0,2 ml of protoplasting buffer
   Incubate at 37°C for 1-2 hours with occasional inversion of the tube to prevent sedimentation of the cells
4. Add 0,2 ml of lysis solution
   Mix gently by inversion
5. Incubate at 65°C for 20 min
   Chill rapidly on ice
6. Add 0,2 ml of 5 M potassium acetate (pH 5,4)
   Mix gently by inversion
   Incubate on ice for 15 min
7. Centrifuge for 3 min at 13000 rpm
   Transfer supernatant to a fresh tube
8. Add 0,6 vol of isopropanol
   Mix gently by inversion
   Let stand at room temperature for 5 min
9. Centrifuge for 30 seconds at 13000 rpm
   Discard supernatant
10. Add 1 ml of 70% Ethanol
    Mix by inversion
    Let stand at room temperature for at least 10 min
11. Centrifuge for 30 seconds at 13000 rpm
    Discard supernatant
12. Dry pellet briefly
    Dissolve DNA in 20-50 µl of TE buffer

2 ml YPD tubes
or
8 ml minimal medium supplemented with 50 mM sodium phosphate pH 6,4

Sterile water

Protoplasting buffer

• 10 µl/ml 2-mercaptoethanol
• 100 mM Tris-HCl pH 7,5
• 10 mM EDTA pH 7,5
• 4 mg/ml Yeast Lytic enzyme (stored at –20°C)

Lysis solution

• 0.2 M NaOH
• 10 g/l SDS (= 1%)

5 M potassium acetate (pH 5,4)

Isopropanol

70% Ethanol

TE buffer

• 10 mM Tris-HCl pH 7,5

1 mM EDTA