Difference between revisions of "Filamentous fungi genomic DNA isolation"
Revision as of 12:41, 9 November 2006
Genomic DNA prep from filamentous fungi like Neurospora.
Note: this is incomplete, some verifications and clarifications needed before it will be considered finished.
- Lysis buffer:
- 50 mM Tris-HCL
- 50 mM EDTA
- 3% SDS
- 1% 2-mercaptoethanol (add just before use)
- Protenase K
- Chloroform:phenol (1:1)
- SEVAG (chloroform:isoamyl alcohol, 24:1)
- 3 M NaOAc (ph 8.0)
- Ethanol (100%, -20 °C)
- Fill a 1.5 mL eppendorf microcentrifuge tube 2/3 to the joint with group lyophilized mycelium (60-100 mg dry, or 0.5-1.0 g wet, ground in in liquid nitrogen)
- Alternatively you can grind 1 g of dried (vacuum filter mycelium first) in a mortar and pestle treating with liquid nitrogen 5-6 times. our the frozen powder into the eppendorf tube.
- Note: you only want to process about 1g in each eppendorf tube, if there is more than this, split to two separate tubes.
- Add 660-750μL + 10 μL Β-mercaptor.
- Vortex to insure good mixing of solution, incubate in 65 °C water bath for 1hr.
- Spin down to remove cell debris. 5min at 3400 RPM. Transfer aqueous phase (top) to 1.mL eppendorf. Do not take any of the cellular debris from the interface. Don't be greedy this prep extracts a lot of DNA.
- Add 700 μL of SEVAG; (adjust volume if needed to meet a 1:1 ratio of SEVAGE and aqueous phase) and vortex. Microcentrofuge at 12,000 g for 10 min.
- Note: some people top off with EB buffer so that total volume is so that volume is equal among the samples for spinning. Be careful as sometimes tops are loosened by chloroform.
- Transfer aqueous phase (top) to new tube.
- Note: these last two steps (SEVAG, spin, transfer) can be repeated to insure cleaner DNA prep depending on your needs and pipette techniques.
- Remove aqueous phase (top) to a new tube (approx. 550-600 μL). Add 20 μL of 3 M NaOAc. Top off with isopropanol. Invert gently. You should see DNA "ropes" precipitating.
- Microcentrifuge for 2 min. Pour off supernatent (top layer). Invert tube for 1 min to dry.
- Add 300 μL EB (or ddH20) and 1 μL of 100 mg/mL RNAse and place in 65°C for 10-15 min. Finger vortex (you don't want to shear this nice long DNA now do you?).
- (Optional) further cleanup with the PEG DNA protocol OR
- 250 μL 7.5M Ammonium acetate. Spin at max speed 5min to pellet protein debris. Take supernatent out, add 750 isopropanol.
- Microcentrifuge 30s to 2 min to pellet the DNA. Pour off the supernatent and rinse the pellet with 100% EtOH. Wash again with 70% EtOH.
- Dry tubes in vacuum oven at 50°C for 15 min at most or dry in speed vac.
- Resuspend in pH 8 TE buffer.
Initially prepared by *Stajich 14:41, 9 November 2006 (EST)
Relevant papers and books
- Sambrook and Russell Molecular cloning manual.