DNA purification from agarose gels
KEITHH at MAINE.MAINE.EDU
Thu Jul 20 07:29:06 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.
You might make it a little bit easier on yourself by increasing the salt
concentration in the running buffer you put it in the well. We used to
do a similar thing years ago, running the DNA into a high salt buffer
(either 1M or 5M NaCl - don't remember which) where it effectively stops
migrating. The principle is the same as salt gradient sequencing gels.
Increasing the local salt concentration reduces the resistance, thereby
reducing the voltage drop and slowing the migration of the DNA. Anyhow,
you may not have to monitor your DNA quite so closely to make sure it
doesn't migrate beyond the well.
Thanks for the post because it reminded me of trying this way of gel
purifying bands. I've been having trouble recently getting >14kb
fragments out of gels using either QiaEx or NA45 paper.
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