Fwd: Re: Genomic DNA extraction

Graham Thomas gxt5 at psu.edu
Thu Aug 1 17:11:12 EST 2002


>Andrea,

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 
precipitate

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)


cheers!
Graham

>  > 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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