DNA purification from agarose gels
seidel at mendel.Berkeley.EDU
Mon Jul 24 02:04:07 EST 1995
: >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.
: Keith Hutchison
Not to be a stickler for details, but as far as the "principle of salt
gradient sequencing gels" goes, my understanding is that by increasing
the salt, and thus the conductance, the voltage drop doesn't change,
as the voltage represents a potential between two points and is not
dependent on resistance, however, the electric field IS inversely
proportional to the conductance. Thus by reducing the resistance,
the electric field is reduced, and thus a charged particle, i.e. DNA,
feels less motive force although the voltage is unchanged.
(seidel at mendel.berkeley.edu)
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