Isolation of yeast gDNA

  1. Collect the cells cultured overnight in an EP tube. Cell volume is 100uL.
  2. Add 100uL STES buffer, mix and spin down, and discard the supernatant to remove media.
  3. STES buffer contains 0.5M NaCl, 0.2M Tris-HCl pH 7.5, 0.01M EDTA, and 1% SDS.
  4. Add 100uL STES buffer and 100uL acid washed 0.4mm glass beads.
  5. Mix well before vortexing. If not mixed well, cells may not break efficiently.
  6. Vortex for 3min with multiple microtube mixer (TOMY).
  7. Add 200uL TE, pH 8.0, and 200uL phenol/chloroform/isoamyl alcohol, pH 8.0.
  8. For RNA isolation, use pH 4.0-5.0 phenol/chloroform.
  9. For genomic DNA isolation, use pH 7.0-8.0 phenol/chloroform/isoamyl alcohol.
  10. Vortex for 3min with TOMY to mix the phenol layer and broken cell layer.
  11. Centrifuge at 4C for 5-10min and transfer the uppermost supernatant, TE layer 200uL, to a new EP tube.
  12. Avoid taking the phenol layer because phenol inhibits enzyme activity during PCR.
  13. Add 20uL 3M Sodium Acetate, 0.1X volume of TE buffer.
  14. Sodium acetate provides a positive charge center to help DNA/RNA precipitate.
  15. Add 500uL 100% EtOH, 2.5X volume of TE buffer.
  16. Incubate at -70C for 15min or at -20C for 30min-1hr, up to overnight.
  17. Centrifuge at 4C for 10min and discard the supernatant.
  18. Add 1mL 70% EtOH and invert at RT for 10min to remove salts.
  19. Use 70% EtOH because more diluted EtOH can dissolve DNA/RNA.