Purifying small amounts of DNA for PCR

Tim Jackson 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
consistantly.

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.

good luck!

tim

Mikael Niku wrote:

> Hello!
>
> 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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