Yeast large DNA prep (wet cell volume: 5mL)

  1. Culture 200mL yeast cells in YPD from OD 0.3 to log phase.
  2. Culture for about 10-12hr.
  3. Do not culture overnight.
  4. Spin down cells in 50mL conical tube at 4C, 4000rpm, for 5min.
  5. Resuspend and fill up to 20mL with sterile distilled water.
  6. Centrifuge at 4C, 4000rpm, for 5min and decant off water.
  7. Resuspend and fill up to 20mL with DW, then add 500uL 2-mercaptoethanol.
  8. Incubate at RT for 15min with rotator.
  9. Centrifuge at 4C, 4000rpm, for 5min, then pour off supernatant.
  10. Resuspend and fill up to 10mL with sorbitol buffer.
  11. Sorbitol buffer is 1M sorbitol distilled and 0.01M EDTA, pH 8.0.
  12. Add 500uL zymolase, 10KU/mL in TE, 5kU. Store at -20C and move to 4C the day before use.
  13. Incubate at 37C for 1hr using rotator or shaking incubator.
  14. Test viscosity by SDS.
  15. Move a small amount of cell to 7mL microtube and add SDS 1:1 to check whether it becomes viscous.
  16. Add SDS to final concentration 1% and incubate 30min at 65C.
  17. Optionally add 100uL Protease K.
  18. Protease K condition: Sigma 10mg/mL, final 250ug/mL; typical working concentration is 50-100ug/mL.
  19. Optionally incubate at 37C for 1hr.