Fungal DNA Isolation

  1. Grow fungus in solid medium (media and time depend on fungus)
  2. Collect 5 plugs using cork # 3 and place them in a 1.5 mL microfuge tube
  3. Add one/two (depends on fungus) little scoops of glass beads (425-600 microns; Sigma G8772)
  4. Shake tube using the Silamat S5 for 10 seconds (or Disruptor Genie for 2 minutes)
  5. Add 450µL Buffer H (below) and repeat step 4
  6. Place at 37°C for 30 minutes
  7. Centrifuge samples for a few seconds in the small black centrifuge
  8. Add 450µL Tris saturated Phenol and shake in your hands until mixed
  9. Centrifuge samples for 10 minutes and transfer supernatant (upper layer) to a “phase lock gel tube”
  10. Treat with 20µL of Rnase (20mg/mL) at 37°C for 30 minutes
  11. Add 450 µL of Phenol:Chlorophorm:Isoamyl and shake in your hands until mixed
  12. Centrifuge samples for 10 minutes and transfer supernatant (upper layer) to a 1.5 mL or 2 mL microcentrifuge tube
  13. Add 40µL of 3M NaOAc (.1X volume) and 800µL cold 100% ethanol (2X volume) and place samples at -20°C for 15-30 minutes.
  14. Spin for 10 minutes to pellet
  15. Wash pellet with 70% ethanol and spin for 10 minutes
  16. Let samples air-dry for a few minutes
  17. Resuspended in 200-500 µL autoclaved distilled water by tapping with fingers
  18. Dry in SpeedVac for 20-30 minutes (or 70°C for 10 minutes)

 

Fungal Lysis Buffer:
250µL 8.0M LiCL
800µL 0.25M EDTA
200µL 1M Tris (pH 7.5)
500µL 20% SDS
18.25mL Distilled H2O
20mL