Fungi gDNA prep

  1. Culture fungi in PD 200mL at 25-30C, 125rpm, for 1-2 days.
  2. Transfer culture to 50mL conical tube.
  3. Harvest cells using filter membrane, Miracloth Merck 475855.
  4. Squeeze with fingers to remove moisture.
  5. Spread the membrane and harvest the cells.
  6. Pour liquid nitrogen and grind it.
  7. 막자사발에 옮긴 뒤 액체 질소를 부어 얼리고 간다.
  8. Harvest and transfer to EP tube.
  9. Add STES 400uL and glass beads.
  10. Use mixed bead sizes so fungi can be ground efficiently.
  11. Bead beating at 6000rpm for 15sec x 3 cycles.
  12. Add TE buffer 500uL and transfer to 50mL conical tube.
  13. Add PCI 4mL plus TE 3.5mL and incubate 5min.
  14. Centrifuge 4000rpm at 4C for 30min.
  15. Transfer supernatant to EP tube.
  16. Centrifuge 13,000rpm at 4C for 10min.
  17. Proceed to EtOH precipitation.