genomic DNA isolation kit
JLISSEMORE at JCVAXA.JCU.EDU
Mon Feb 13 14:02:55 EST 1995
About a month or so ago, I requested information from the Worm-net about rapid
genomic DNA preps that might be suitable for an undergraduate lab. I did get
one suggestion about an actual kit from a company called Gentra. They were
kind enough to send a sample kit (enough to isolate DNA from a couple of plates
worth of worms) which I tried out last week. The bottom line is that the
method is very easy (no phenol/CHCl3 extractions!) and it gives a good yield of
DNA of fairly high molecular weight (approx. 50+ kb, based on uncut lambda as a
marker) that cuts well with restriction enzymes. I haven't tried it yet for
PCR or Southerns, but I have been told by a source that requested anonymity
that it works fine on Southerns.
Now for a few details: I estimate I got about 50 ug of DNA (based on
ethidium bromide fluorescence in a gel) from two 60 mm plates of N2s that had
just finished off the last of the lawn. The method still requires a long
digestion with proteinase K in the presence of SDS (3 h to overnight) but the
remaining steps took about an hour to complete, not including time to dry and
redissolve the DNA pellet. After proteinase K treatment, RNase is added,
proteins are removed by salt precipitation, and DNA is precipitated with
isopropanol followed by a 70% ethanol rinse. The salt precipitation step
in the protocol says to vortex at high speed for 20 seconds and I still got DNA
in the 50 kb range; I did not try a gentler mixing step but I assume you could
try something a little gentler than vortexing. The pellet is air dried and
resuspended in TE.
The kit provides all ingredients except for proteinase K, isopropanol,
and ethanol and it includes a very detailed, easy to follow protocol
specifically designed to isolate nematode DNA. The
protocol says the yield is 15-50 ug from 40-90 mg of worms from a 100 mm plate;
I think my yield was toward the high side.
For more info, contact Gentra Systems, Inc. at (800)866-3039 and ask
about the Puregene DNA isolation kit. They were very helpful and were glad to
send a sample kit. I hope some of you find this to be useful information. And
no they aren't giving me any money to pass on this info. After I've tried out
some Southerns and PCR with this DNA I'll post another note.
Many thanks to all of those who replied to my initial request for info!
John Carroll University
University Heights, OH 44118
email: jlissemore at jcvaxa.jcu.edu
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