BustnGrab - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

  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 5 minutes.
  2. Add 200 μl of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0).
  3. Resuspend pellet by vortex or pipette.
  4. Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to in a 95°C water bath for 1 minute. (I)
  5. Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to in a 95°C water bath for 1 minute. (II)
  6. Vortex 30 seconds.
  7. Add 200 μl of chloroform; vortex 2 minutes.
  8. Centrifuge 3 minutes, 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 at -20°C to increase yield.
  11. Centrifuge 5 minutes, room temperature, 20,000 × g. Remove supernatant with a pulled Pasteur pipette by vacuum aspiration.
  12. Wash the pellet with 0.5 ml 70% ethanol, spin down as described in step 8 above. Remove supernatant.
  13. Air-dry the pellets at room temperature or for 5 minutes at 60°C in a vacuum dryer.
  14. Resuspend in 25–50 μl TE [10 mM Tris (pH 8.0), 1 mM EDTA (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 (Ambion) should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).

Harju, S., H. Fedosyuk, and K.R. Peterson. 2004. Rapid isolation of yeast genomic DNA: Bust n’ Grab. BMC Biotechnol. 4:8. link