Purifying small amounts of DNA for PCR
tim.jackson at nrc.ca
Tue May 23 09:30:23 EST 2000
i am currently using the Puregene DNA isolation kit (Gentra Systems:
www.gentra.com) for extracting 5 day old ethanol-fixed fish embryos. i'm
using 1/2 of their "5-10mg fixed tissue" protocol and really goosing up the
proteinase K (5uL of 20mg/mL) but that's mostly because i'm not grinding up
the embryos and need to digest away the egg casing. anyway, this is
working very well for me - i always get visible pellets and my PCRs work
the old tried and true phenol/chloroform is always a pretty damn efficient
procedure. we used that in a forensics lab i worked in and used Microcon
(100's, i think) columns to concentrate/clean the final DNA extract (in
lieu of precipitation) and used to be able to get DNA out of just about any
trace amount of tissue.
Mikael Niku wrote:
> Which method would you recommend for purifying
> small amounts of DNA to be used as a PCR template,
> in order to minimize loss of DNA in the procedure?
> I'm trying to PCR DNA from microdissected histological
> samples, digested with proteinase K. Each sample typically
> consists of a piece of tissue (perhaps 100's or 1000's of cells,
> and I should be able to detect a single-copy gene present
> in only part of them) in 20 ul digestion buffer. My problem is that
> using the crude digest as a template, I'm getting annoying unspecific
> products. These are not produced when using purified DNA as a template,
> so they probably are related to the purity of the template.
> Mikael Niku URL: www.helsinki.fi/~mniku/
> University of Helsinki Dept. Basic Veterinary Sciences
> - Mitäkö mieltä olen länsimaisesta sivistyksestä?
> Minusta se olisi erinomainen ajatus!
> - Gandhi
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