Isolating C. elegans Genomic DNA
from Michael Koelle's southern blot protocol
The volumes in this protocol are for worms harvested from a single 10cm plate. Increase them as needed for more plates.
- Seed 10 cm agarose plates with HB101. Agarose is preferred over agar because it contains less inhibitors of restriction enzymes.
- Harvest worms before the plates starve, around the time they form a wave accross the plates as they finish off the last bacteria.
- Wash and pellet the worms in TEN buffer. Take up the worms in 500µl TEN.
- Per 500µl TEN add:
- 25 µl 10% SDS
- 5 µl Protease K (stock 10 mg/ml)
- 1 µl β-mercaptoethanol
- Incubate for 1 hour at 60°C, inverting occasionally. Then add another 5 µl Protease K. Incubate a second hour. Add more protease K. Incubate a third hour or overnight if there remains too much gloppy worm goop.
- Phenol/chloroform extract with 0.5 ml per starting plate. To increase the yield, re-extract the phenol/chloroform phase with more TEN.
- Precipitate the DNA by adding 2.5 volumes of EtOH. The genomic DNA will form a nice clot, which you can pick out with a pipet tip, or a glass tip from a capillary.
- Resuspend the pellet in 0.5 ml TEN per starting plate. This resuspension is difficult, and may require a 4°C O/N step, followed by 1-2 hours at 50°C.
- Add 3 µl RNAse A (10mg/ml stock) per starting plate. Incubate at 37°C for 1-2 hours.
- Repeat the Phenol/chloroform extraction as in step 6. Then extract once with chloroform only.
- Precipitate the DNA by adding 2 volumes of EtOH. Pick the DNA out as before, and wash in a tube of 70% Ethanol. Let sit for 1 hour at room temp to remove salts.
- Spin for 10 minutes at 13,000 rpm, remove the supernatant, air dry the pellet, and resuspend in TE (about 100 µl per starting plate). Resuspension may again be difficult and require a 4°C O/N step, followed by 1-2 hours at 50°C.
TEN: 20 mM Tris pH 7.5, 50 mM EDTA, 100 mM NaCl