Quick fly DNA prep (was: genomic DNA extraction kits
M.Champagne at cellbio.duke.edu
Tue Jan 27 06:51:11 EST 1998
By popular demand, here's the genomic DNA extraction protocol i've been using.
I've used it successfully with flies, larvae and pupae. I don't know who 1st
wrote the original protocol, but it's been adapted by G.Thomas, K.Edwards and
myself, all from the Kiehart lab at Duke University. This version was optimized
for rapidity/purity, thought my yields were better than the kits or other preps
Quick fly DNA prep
(by M.Champagne, G.Thomas, K.Edwards and who knows who else!)
1. Collect flies, larvae, pupae and freeze them (makes the grinding step
much easier (-: Ex.: 1-10 flies in a 1.5 ml eppendorf for this
i was able to scale this up to 100 flies).
2. Get materials ready for grinding:
65c water bath,
pestle that tightly fits your eppendorfs (we have ours mounted on an engine)
EtOH 100% and 70%
Grind Buffer: 0.2 M Sucrose
0.1 M Tris, pH 9.2
50 mM EDTA
3. Add 120 ul of grind buffer to the flies and homogenize for roughly a
minute until all big pieces are broken. (Don't get bodies trapped under
the point of the pestle, this leads to poor contact around the sides. You
shouldn't be able to identify fly parts)
4. Rinse debris off the pestle into tube with 370 ul more of grind buffer
5. Vortex tube hard for 10 sec. and place immediately in 65c water bath
for 10 min.
6. Add 75 ul 8M KAc and vortex briefly to mix, place on ice at least 15 min.
(up to a few hours is fine).
7. Add 0.5 ml of Phenol/Chloroform (1:1), vortex hard for 30 s.
8. Spin 5 min. Take tube out of centrifuge very gently. (the fly debris
will pellet at the bottom, the white protein junk will be at the
interphase and the
DNA extract will be on top).
9. Transfer the supernatant (top) by pipetting into an eppendorf
containing 1 ml
100% EtOH. Vortex to mix, and leave at room temperature for 4 hours (or
IMPORTANT: Be picky, not greedy - always leave some supernatant at the
interface, else you'll carry over some protein junk. You should handle the
tube gently to avoid mixing the interface/phenol phase with the supernatant.
NOTE: If you need 'very' clean DNA, you might want to repeat the
Phenol/Chloroform step, then followed by a Chloroform step. In my tests,
i've found that i could skip those extra steps and still have great
sensitivity with my PCR assay.
10. Centrifuge the tube 10 min at high speed to pellet DNA and RNA. Dump
EtOH (watch out, the pellet might be loose).
11. Wash pellet with 70% EtOH, spin, remove all liquid from pellet, air
dry about 30 min.
12. Add 50 ul H2O to pellet. (I let it swell 15-20 min. at 20 min., then
resuspend by pipetting; though some claim you need to let the pellet swell
and dissolve it overnight at 4c). Use 1-5 ul per PCR reaction of 30-50
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