Large plasmid inserts, electroporation

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri May 5 15:13:30 EST 2000


In article <8er7uu$44s$1 at nnrp1.deja.com>, ladasky at my-deja.com wrote:
: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.
:
Somewhere in my thesis (which I don't even know where it is), there is
a reference to a paper describing electroporation of 180 kb plasmid 
into E.coli with >10^6/ug efficiency. For very large plasmids, one needs
to use relatively lower field strenght (10-12 kV/cm as opposed to 
15-18) and relativly longer pulses (tau of ~ 10 ms as opposed to ~5). 
This is documented somehwere in NAR (~ 1995-7) and is probably due
to partial DNA denaturation in high EF that leads to random annealing
and DNA "clotting" as described somewhere in Biomembranes and 
Bioenergetics (if I am not mistaken, the denaturation is due to Vin's 
(sp?) effect). 

        - Dima



        - Dima




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