aberrant DNA migration on an agarose gel

teresa_mogul at my-deja.com teresa_mogul at my-deja.com
Tue Jul 25 00:40:15 EST 2000



Hi Scott

It is possible that during your washing, centrifuging and drying you are
damaging your DNA. ie putting little nicks in it. This would mean that
you have your target DNA and much smaller fragments of different
intensity depending on the amount of damage.


 Scott Greene <sgreene at biochem.dental.upenn.edu> wrote:
> Hi.
>
> I've noticed an issue with DNA migration that has me a bit puzzled;
> perhaps someone here has seen the same thing happen or has a thought
as
> to what may be occurring:
>
> I cut plasmid DNA with a restriction enzyme and then run the digestion
on
> an agarose gel to separate the various bands that result from the
> digestion (in my case, only 2 well separated bands result).
>
> I then *cut* the desired band out of the gel with a razor blade and
follow
> the electroelution procedure using a dialysis membrane.  The agarose
slice
> with DNA is placed into this dialysis membrane and subjected to 400
volts
> for about 10 minutes, which pushes the DNA out of the agarose slice
and
> onto the wall of the dialysis membrane.  I reverse the polarity for a
few
> seconds to push the DNA off the wall of the membrane and into the TBE
> buffer, where it can be collected.  Note that I'm following the
movement
> of DNA using ethidium bromide.
>
> After collecting the DNA, I precipitate it with 1/10 volume of 3 molar
> sodium acetate (NaOAc) and 2 volumes of absolute ethanol.  This
solution
> is placed into the freezer overnight.
>
> The next morning, I centrifuge the solution, remove the supernatant,
wash
> the DNA pellet with 70% ethanol, centrifuge again, and then pour off
the
> supernatant.  I dry the pellet using centrifugation under vacuum
> ("Speedvac"), resuspend it in a small volume of dH20 (maybe 15
> microliters) and then take maybe 1 or 2 microliters to run an agarose
gel
> to check the DNA.
>
> I find that I routinely see two bands in the gel lane.  These
> multiple bands are of different intensities.  One band is the size I
> would expect following the electroelution procedure.  The other band
> (ofter much more intense) is a band that migrates very fast, often
below
> the 2-kb band of the lambda-hindIII marker (which is puzzling because
the
> expected size of the band might be around 5 or 6 kb).
>
> I've seen this happen repeatedly.  I've ruled out contamination on the
> razor blade because I always use a new one to cut the DNA out of the
agarose.
>
> Does this ring a bell with anyone?  Any explanations for the aberrant,
> fast migration of the DNA?
>
> Thank you,
> Scott
> sgreene at biochem.dental.upenn.edu
>
>


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