Yeast DNA extraction

Materials needed:

Procedure:

  1. Prepare 1.5ml screw-cap tubes (1 per sample):
    1. 0.4 g glass beads (corresponding to a volume of roughly 150 ul)
    2. 200 ul DNA extraction buffer
    3. 200 ul Phenol:Chloroform:Isoamyl alcohol
    4. Yeast cells (using a pipette tip, take a big chunk of cells; it is best if the cells are fresh)
    5. Label tubes
  2. Close screw caps tightly
  3. Load onto FastPrep (let someone show you how)
  4. Run the S. cerevisiae extraction protocol
  5. Take out tubes
  6. Spin down briefly in small tabletop centrifuge for a few seconds
  7. Add 200 ul of 1x TE
  8. Centrifuge at top speed for 5 min at room temperature
  9. After centrifugation, samples should contain an aqueous phase (top, containing the DNA), an interphase (white), and an organic phase (lipids, proteins, etc.)
  10. Take a clean 1.5 ml tube (no need for screw caps) for each sample, label, and add 1 ml 100% ethanol
  11. Transfer the aqueous phase of each sample (from step 8) to the new tubes containing 100% ethanol
  12. Invert samples a few times (should see nucleic acid precipitation (cloudy))
  13. Spin at full speed for 5 min (should see nucleic acid pellet)
  14. Discard the supernatant, keep the pellet
  15. Add 500 ul of 70% ethanol to wash the DNA pellet
  16. Centrifuge at top speed for 5 min
  17. Remove as much of the supernatant as you can, leaving as little water-ethanol as possible
  18. Add 50 ul 1x TE
  19. Recommended: add 10 ug/ml RNase (a 1/1000 dilution of 10 mg/ml stock RNase)
  20. Recommended (continued): at 37 C for 5 min

Author: Enrico Tenaglia
September 2018