Separation of DNA fragments.

Dennis J. Templeton djt2 at po.CWRU.Edu
Fri Jul 31 10:43:43 EST 1992


In a previous article, jaf at bio.aau.dk () says:

>How can two DNA fragments  of MW 3300 kb and 3400 kb be separated on an agarose
>gel?? They have to be separated so much that one can isolate them without
>contamination. 
>

jaf-  I have to disagree with a posted followup that says to use a 4% 
gel; this would work with a smaller fragment but not with 3.4k.

We have had good luck by digesting the set with an enzyme that cuts in the
middle of the piece you *don't* want, effectively removing it from the
piece you do.  It's surprising how often a good enyme for this can be
found.

Often too, judicious use of CIP can remove the effects of a small level of
contamination... for example, are you preparing a vector for cloning?  then
CIP the mix and any contaminating fragment should be unligatable.

My rule of thumb is that it is difficult to resolve pieces that differ by
less than 10%.  Also, contamination seems to smear *up* the gel.  If you
are separating pieces of 4kb and 3 kb, the 4 kb will be contaminated with
some of the 3kb, but the 3 kb will be much purer; the front running band is
cleaner (in my experience)

good luck,

dennis



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