Marcus Schmidt wrote:
> > 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.
> > 59:687).
>> Gee, and I thought Ogram et al. (1988) had been the first.
My apologies. I should have written "for extracting DNA from native
bacteria in sediments and soil *for PCR*...". At the time we first
presented the method in Abstracts of the ASM in 1992, no one (that I
know of) had published a method on the successful amplification of DNA
from native bacteria in soil or sediments. Others later that year (e.g.
in Stackebrandt's lab in Australia) were successful, as well. But of
course we relied on the pioneering work of Ogram et al., Steffan and
Atlas, Holben et al., Tsai and Olson, and others.
>> 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.
Many thanks! We actually tested the beadbeater lysis method (versus our
previous one using freeze/thaw, SDS, lysozyme) using Bacillus endospores
and found the beadbeating method (first used by Ogram et al., I believe)
to be far superior both in lysis efficiency and in DNA yield (More' et
al. 1994. ibid.)
> It's not. No alternative published - so far, with the possible exemption
> of sonification, which, however, shears the DNA even more.
With regard to DNA shearing, Dan Miller -- a former colleague of mine in
the Ghiorse lab at Cornell -- found that shearing was not a problem if
the mini-beadbeater was run at less than 3500 rpm. This can be a problem
as the newer beadbeater (manufactured by BioSpec products) will not run
at that speed. Our solution has been to purchase the larger 8-tube
jherrick at lanl.gov
Microbial Ecology and Genetics
Environmental Research and Development
University of California/Los Alamos National Laboratory