Fwd: Re: Genomic DNA extraction
gxt5 at psu.edu
Thu Aug 1 17:11:12 EST 2002
I didn't catch the original posting here, but your sds/sucrose/edta
protocol looks like a modification of a protocol that we routinely
use for small numbers of flies (up to 10 in a 1.5ml tube) that nets
about 0.5ug/male ~1ug /female:
[Unfortunately I do not know who to attribute this too as it is not
listed on the protocol I was handed (and still use!) in 1984]
Using a motorized rotary pestle we grid directly in the
Sds/sucrose/edta/tris (125ul) until the flies are well 'dispersed'
(30-60s). This is made up to 500ul with an additional 375ul of buffer
which we rinse the pestle off with to maximise recovery.
This is heated 65C for 10min
Next add 75ul of 8M potassium acetate and a lot of junk and sds will
incubate in ice for 15 mins
spin 5' full speed in ufuge. Repeat (the pellet is not terribly firm
and some stuff sometimes floats)
add 1ml of 100% ethanol AT ROOM TEMP. Give it a few mins to fully ppt.
spin at room temp for 10 mins.
rinse three times with 70% ethanol, dry and resuspend in te
this is simple fast and usually digests pretty well (include some
RNase A in the digestion and 4mM (Na) spermidine may help too)
> > Hallo,
>> I am struggling with the isolation of genomic DNA of Drosophila. First I
>> tried a tissue kit which worked well for marine organisms but did not
>> work for Drosophila. Next I tried a protocol using frozen flies,
>> grinding them in a buffer containing SDS, Sucrose and EDTA, heating,
>> Phenol-Chloroform extraction and final EtOH precipitation. The DNA yield
>> was poor too, only 140 ng from 15 flies. For a southern blot experiment
>> I need much more genomic DNA.
>> I would be glad if somebody could send me a protocol, which works well.
>> Thanks a lot, Andrea.
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