On Wed, 20 Apr 2005 20:00:33 +0100, Tanya Kuritz wrote:
> <font size=3>I see several problems with the study design, in the first
> place. Isolating an intact, circular DNA of several hundred Kb is
> not trivial. One has to embed the organism first and then digest,
> and/or leach, other molecules that may interfere with digesting.
> There are several methods to do so, and I can send refs including mine,
> on how to do so. Most of this literature was published back in the
> early 90-s, when pulse-field GE of microbial DNA was hot.<br><br>
> Another problem is enzyme selection. Rare cutters do not
> necessarily cut rarely, or cut at all. In my experience, PstI (a
> frequent cutter for many systems) was an extremely rare cutter for
> cyanobacterial genomic DNA. It is worh doing some bioinformatics
> search first, rather than guess. Evolution worked for too long, and
> we have too little time and knowledge to rely on guessing.<br><br>
Thank you for your suggestions.
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.
Thank's, The Duck.