Concatemerized Plasmid DNA

Geoffrey Kidd 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
Aptagen, Inc.
www.aptagen.com

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
> reliable?
>
> Any help is much appreciated.
>
> Regards,
>
> _______________________________________
> Antonin Tutter
> Salk Institute for Biological Studies
> RBIO-J
> 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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