From OpenWetWare
Jump to navigationJump to search

Home        Research        Lab Members        Publications        Software        Internal       

Rather Rapid 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 (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0), 200 µl 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 µl 4M ammonium acetate, mix, and then add 1 ml 100% EtOH and mix.
  10. Spin for 2 minutes and dry pellet. Resuspend in 50 µl TE and use 10 µl per digest.