aberrant DNA migration on an agarose gel

Kristine E Gouveia Kristine.Gouveia at bms.com
Tue Aug 1 15:17:17 EST 2000


The problem you are having is due to exposing the DNA to UV light in the
presence of EtBr.  Small nicks are introduced into the DNA.  These nicks will
alter the migration of the DNA.  That is why you are seeing two bands.  You
also are probably finding that cloning is a problem.  What I have been taught to
do is to run a small sample of the DNA (like 5ul) in a lane next to the larger
sample that you will excise the band from.  When the gel is done running, cut
that
single lane out, and stain it in EtBr.  Expose that lane to UV and mark off where

your band is that you want to cut out.  Transfer that lane back to the original
gel, making sure it goes in the same way that it came out.  Now cut out the area
of
the gel next to where you marked off on the small lane.  You will now be able to
gel purify you band using the dialysis method.  The only thing is that you will
not be able to track the migration of the DNA out of the gel since it doesn't
contain
any EtBr.  Just run it for as long as you think it should take.  I usually run it
at 100V for about an hour.  I hope this solves your problem.  I know it helped me
with my cloning.
Good Luck
KG


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