On Wed, 20 Apr 2005 22:00:27 +0200, The Duck <theduck at earth.com>
wrote:
>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
years.
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!)
regards,
bob
