Large plasmid inserts, electroporation

ladasky at my-deja.com ladasky at my-deja.com
Thu May 4 02:13:44 EST 2000


In article <3910F14D.67D24DC0 at gpu.srv.ualberta.ca>,
  wgallin at gpu.srv.ualberta.ca wrote:

> At 05:32 PM 5/3/00 +0100, yan wu wrote:
> >I am looking for plasmid or phagemid vector(s) which
> >can hold 50kb DNA insert. I
> >appreciate it very much if anybody can send me
> >information about the name of the plasmid
> >and the place I can get it. Thank you very much. My
>
> For that size people have typically used cosmids.  The only difference
> between cosmids and plasmids is the inclusion of the lambda cos site
> in the vector, allowing packaging into a lambda phage particle for
> easy transfection of the bacteria.  The high transformation rate is
> mainly important for making libraries.
> However, why not clone into your plasmid of choice and electroporate
> to transfect your bacteria.  It may not be the most efficient method,
> but if you are not making libraries it probably won't matter.
>     As far as I know there is nothing special about plasmids that
> prevents them from holding a big insert.  The reason people haven't
> used them for that, historically, is that the chemical transfections
> are size-limiting.  I don't think electroporation is.
>
> Warren Gallin

I can corroborate part of Warren's remarks.  I was going through some
old magazines recently, and came across a 1983 Scientific American
article on plasmids.  The article described several naturally-occurring
plasmids in E. coli whose sizes had been determined by restriction
mapping, and were on the order of 80 to 100 kB.

--
John J. Ladasky Jr., Ph.D.
Department of Structural Biology
Stanford University Medical Center
Stanford, CA 94305


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