DNA purification from agarose gels

David Micklem drm21 at mole.bio.cam.ac.uk
Fri Jul 21 14:55:46 EST 1995


> In article <3uj8ko$eia at news.acns.nwu.edu>, andy chien
> <tubeman at merle.acns.nwu.edu> says:
> >
> >Our lab does not use any of these kits for the purification of
> >DNA fragments from gels.  Instead, we use a technique that was
> >recently described in Biotechniques.  What we do is we run our
> >DNA sample, at 50-100 mV for a 10 cm gel, and as soon as the
> >band is well resolved, we use a sterile razor blade or scalpel
> >to carve out a  well immediately in front of the band of
> >interest.  The closer the well is to the band, the easier it
> >will be to isolate.  The well is usually about 1 cm. long.
> >Then we lower the buffer in the running chamber so that it is
> >just below the level of the gel, and not spilling over the top.
> > Then we fill the newly-cut well with 200-400 ul of running
> >buffer, and crank the gel at 150 mV for 1-2 min.  Then we pipet
> >out the band, which has run into the well, which we can also
> >track by UV.
> 

Surely that would be Volts, not mV...

Anyway, another varient of this technique is (IMHO) even easier, and gives
you your DNA is a smaller volume.

Instead of carving out a well, just slice a slit in front of the band you
want.  Into the slit place a small piece of Watman GF/C filter backed by a
single thickness of dialysis tubing.  Adjust buffer layer and run gel as
above.  The DNA migrates onto the GF/C filter, but can't run through it
due to the dialysis tubing.  This method differs from the use of eg NA45
paper in that the DNA is NOT bound to the GF/C paper, so there is no
difficulty in 'eluting' it.
Take out GF/C-dialysis tubing and place in a  500ul eppendorf tube with a
hole in the bottom.  Place 500ul tube in 1.5ml and spin for two minutes.
(For best yield, spin slowly, then add 50ul TE to the GF/C paper and spin
at high speed).
The liquid that comes out can be used directly for ligation, or if you are
paranoid/use crummy agarose then phenol/chloroform/precipitated

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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