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Yeast Genomic DNA Isolation

David Amberg

  1. Grow 100 ml culture to about 1x10 to the 8th
  2. Spin 4 x 15 mls of culture down about 3k x 3 min.
  3. Resuspend entire pool in about 6 mls ddH2O, aliqoute into 1.5ml microfuge tubes and spin down.
  4. Aspirate off super and vortex pellet till suspended.
  5. Add 200ul soln A, add 200ul Phenol/CHCl3 and 0.3g acid washed glass beads.
  6. Vortex 2 min, add 200ul TEpH8 and spin at max speed for 5 min.
  7. Transfer super to a new tube and add 1ml 100% EtOH, mix by inversion.
  8. Spin 2 min, pour off super and recon in 0.4ml TEpH8 + 30ug/ml RNaseA.
  9. Incubate 5 min x 37¡C, add 18ul 5M NH4OAc and 1ml 100% EtOH and store at -20¡C for a few hrs.
  10. Spin down DNA 10 min and recon each pellet in 25-50ul or whatever convenient volume.
  • Solution A:
  • 200ul neat or 2ml 10% Triton X-100 (2%)
  • 1ml 10% SDS (1%)
  • 200ul 5M NaCl (0.1M)
  • 100ul 1M Tris pH8 (0.01M)
  • 20 ul 0.5M EDTA (1mM)
  • Water to 10ml