DNA ligation

killer yeast vvsvetlov at utmem1.utmem.edu
Fri Jul 19 00:14:39 EST 1996


In article <drm21-1807962203360001 at nntp-serv.cam.ac.uk>,
drm21 at mole.bio.cam.ac.uk (David Micklem) wrote:


> >Purification of DNA from the agarose gels using DE81 paper
> >
> You can get this to work?!  I tried (ages ago) and could get small
> fragments out OKish, at rather poor yield.  Big bits just stuck to the
> DE81. Permanently. Zero yield. Several times.

This particular technique is a part of old Korean martial art that I
learned from the master Heiu-Dong Park. Since I don't like dialysis bags
and all I started using this one, now I have more followers including
secret ones judging by depletion of my buffers and cut filters. This
method does work as described with good yield on fragments up to 11 kb
(pEG202 derivatives), I've been using it for four years without any
problem, mostly to isolate PCR products (0.1 - 1.5 kb) but all sorts of
digests too including plasmid backbones as above. Overall I've made about
200 constructs using plasmid backbones and inserts isolated using this
technique. Moreover on occasion a bigger fragment (`3 kb) was isolated
using DE81, cut with another enzyme, run on the gel and one of the
resulting bands (~2 kb) was isolated again with DE81 and cloned, so at
least it works twice in a row without subcloning. I would find meself in
very peculiar position recommending stuff that does not work. I don't know
how this DEAE paper behaves with fragments larger than 11 kb but within
these limits it has been most reliable method I've used. If one chance
uses a stronger anion exchanger or elutes at low ionic strength I'd expect
problem with recovery. 
Regards,
Vlad



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