how to isolate a large plasmid

clive tregaskes clive.tregaskes at bbsrc.ac.uk
Tue Jun 8 11:57:54 EST 1999


root at titus.u-strasbg.fr wrote:
> 
> who has experience in isolating a large plasmid? I try to isolate a
> plasmid pBluescript SK(+) containing a about 18kb insert but fail.


I've managed to grow inserts over 20kb in pBluescript but also found
problems when isolating them, all I got was genomic DNA.  Cured this by
just growing the bugs  in 25ug/ml ampicillin rather than 100 and got a
decent yield.  I suspect (although if anyone has another interpretation
I'm willing to listen) that you're selecting for plasmids with some
mutation that makes them lower copy number, hence produce less
beta-lactamase and so the bugs are always on the edge at high amp
concentrations
hope this helps

Clive Tregaskes
Institute for Animal Health, Compton, Uk



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