DNA isolation from mud/soil
marcus.schmidt at tu-bs.de
Tue Oct 21 03:50:36 EST 1997
James Herrick wrote:
> In article <pjc-1310971402170001 at 188.8.131.52>, pjc at waikato.ac.nz
> (Peter J Charlton) wrote:
> > Would anyone like to tell me their favourite method and why? or perhaps
> > point me in the direction of a recent review of soil extraction methods?
> Well, I'm about to tell you that ours is the best! No, really, there are a
> lot of good methods out there and they seem to be converging. We published
> one of the first methods specifically for extracting DNA from *native*
> bacteria (as opposed to those added in; a significant hurdle, we found) in
> sediments and soil (Herrick et al. 1993. Appl. Environ. Microbiol.
Gee, and I thought Ogram et al. (1988) had been the first.
> Since that time we have modified the method somewhat and
> successfully tested it on some of the nastiest, organic-rich sediments we
> could find, looking at the same time at the efficiency of lysis of the
> native cells (More' et al. 1994. AEM 60: 1572-1580). The most up-to-date
> published version of this method is in an excellent little book called
> 'PCR:Essential Techniques', edited by Julian F. Burke (1996. Bios Sci.
> Publ., Oxford). I can also send you a fuller version of this protocol via
> e-mail, if you wish.
An important paper of yours, and you have developed a fine method! I
agree with you in the absolute necessity of bead beating - part of my
experimental work had been devoted to the detection of Bacillus
endospores, and bead beating proved to be the only way to crack them up.
> I like our method because it's versatile, reproducible, very fast, and
> it's been used successfully in a lot of labs. The one drawback is that it
> requires a beadbeater for physical lysis of cells.
It's not. No alternative published - so far, with the possible exemption
of sonification, which, however, shears the DNA even more.
One thing that might be worth trying (I didn't do it) is the
PEG-precipitation of purified extracts (I used Wizard instead of
spin-bind, with not fully satisfactory results) suggested by Purdy et al
(1996, AEM 62(10):1905-7). If you did, or anybody else following the
discussion, please tell me. The method is cheap, available, and should
be able to effect additional humic acid removal.
(marcus.schmidt at tu-bs.de)
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