UV damage and cloning, was Re: aberrant DNA migration on an agarose gel

[Michael L. Sullivan]

>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
(lots cut out)

An alternative (and I think easier) approach is to go ahead and run the gel
with EtBr and then make sure to visualize with a UV source that won't
damage the DNA, e.g. a long-wave hand held UV lamp is what I use.  I've
used this approach for years without any problems cloning.  Of course I'm
still careful to try to minimize the UV exposure, but I think the
EtBr-stained DNA can still take a lot of long wave UV without damage.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


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