Rapid Yeast Genomic Prep

Hoffman and Winston, Gene, 1987

  1. Grow 5 ml yeast cultures to saturation.

  2. Collect cells by centrifugation and resuspend in 0.5 ml of water. Transfer cells to 1.5 ml microfuge tube and collect by a 5 second spin.

  3. Pour off supernatant and briefly vortex to resuspend cells in residual liquid.

  4. Add 0.2 ml of Buffer A, 200 ul glass beads, and 0.2 ml phenol:chloroform:isoamyl alcohol (25:24:1).

  5. Vortex 3 minutes (setting #7 on a foam multi-tube vortex adaptor). Add 0.2 ml TE.

  6. Spin 5 minutes. Transfer aqueous to new tube. (OPTIONAL:) Do a chloroform extraction.

  7. Add 1 ml 100% EtOH (RT; cold EtOH will create a large, dirty pellet), invert tube to mix, and spin 2 minutes.

  8. Discard supernatant, and resuspend pellet in 0.4 ml TE (no need to dry pellet).

  9. Add 10 ul 4 M ammonium acetate, mix, and then add 1 ml 100% EtOH and mix.

  10. Spin for 2 minutes and dry pellet. Resuspend in 50 ul TE.

  11. Can now be used for transformation into E. coli for plasmid rescue or in a digestion for Southerns, etc.


BUFFER A
(2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0)

TO MAKE 100 ml:

  • 1.0 ml 1M Tris, pH 8.0 stock solution
  • 1.0 g SDS, or 10 ml 10% SDS stock solution
  • 2 ml 5 M NaCl stock solution
  • 200 ul 0.5 M EDTA stock solution
  • 2 ml Triton X-100