cutting large circular DNA molecules

The Duck theduck at earth.com
Thu Apr 21 13:34:48 EST 2005


On Wed, 20 Apr 2005 21:23:54 -0700, Bob wrote:
 
> 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

I had a chance today to checkout the New England Biolabs catalog (the
online version). Well, that saved me quite some additional work and
answered a number of other questions to, since they have done the
bioinformatics about which enzymes cut prokaryotic DNA rarest pretty well
as Tanya suggested earlier in this thread.
I found a couple of papers were they dealt with intact prokaryotic
chromosomes and they all report that large circular DNA just doesn't enter
the gel. On second thought that isn't really that surprising considering
how PFGE works (switch time related to reorientation time of the
molecules). I was just hoping that I overlooked something and that there
still might be a way to do it (like radically different running conditions
as compared to linear molecules). Your suggestion of contacting the
supplier (Bio-Rad in my case) probably cannot harm, so I will try that as
well. However, I have very bad experience with Bio-Rad and don't really
expect much from them...they are just interested in selling their products
and the technicians are in my experience not very competent when it's
crunch-time (no offense intended.....just my experience).

Thanks bob, The Duck.



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