cutting large circular DNA molecules

Bob bbx107 at excite.com
Wed Apr 20 22:23:54 EST 2005

On Wed, 20 Apr 2005 22:00:27 +0200, The Duck <theduck at earth.com>

>The problem is not so much the isolation, that is in fact fairly easy by
>embedding the guys in agarose. The point just is that it seems to be
>impossible to separate large circular DNA molecules on a PFGE gel, they
>do not even enter the gel but stay in the wells. So my next idea was to
>use restriction enzymes. By now I get bands with the ones I tried (e.g.,
>Sse 8387 I-8-bp cutter) but the fragments are still too small. However, as
>I said in my initial post, I really just want to cut them once so that I
>can separate them using PFGE.

Know what? I think you should talk with someone expert in PFGE, esp of
DNA. That might be the supplier of your equipment. I know the general
issues, but haven’t paid much attention to developments for a few

The issue of how circular DNA behaves in electrophoresis, including
PFGE, will be somewhat different, because the polymer has a different
shape. (Do you know your circles are "relaxed".) Sounds like getting
some expertise on this would really be better than dealing with the
cutting. It may also be "interesting". I wouldn’t be surprised if a
softer gel was called for.

(It is sometimes good to ask what it is you really want to know!)



More information about the Microbio mailing list

Send comments to us at biosci-help [At] net.bio.net