Concatemerized Plasmid DNA
gkidd at aptagen.com
Wed Nov 11 15:11:41 EST 1998
Is it possible that the dimer arose during ligation? Has it ever been shown to
be a monomer?
Geoffrey Kidd, Ph.D.
Technical Services Manager
Antonin Tutter wrote:
> I have encountered the problem of concatemerized plasmid DNA in recent
> preparations of DNA. Nearly all the DNA is in the form of supercoiled
> dimers. I conclude this because migration of uncut DNA is twice that
> predicted for supercoiled, and migrates between linear and nicked when
> partially digested with a unique cutter yielding 50% linear and 50% nicked.
> I believe it is a problem with the cells. The DNA is introduced by heat
> shock and grown in DH5 alphas, which are rec-, but the cells have been
> perpetuated in the lab for some time. So, how can this be happening? Could
> the cells have become contaminated with a rec containing plasmid? Is there
> any selective pressure on the cells to maintain a copy of the rec gene?
> Which takes me to the next logical question, what are some experiences with
> commercially available competant cells? Are they consistently competant and
> Any help is much appreciated.
> Antonin Tutter
> Salk Institute for Biological Studies
> 10010 N. Torrey Pines Rd.
> La Jolla, CA 92037
> email: atutter at aim.salk.edu
> web: http://www-biology.ucsd.edu/~atutter/
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