how to isolate a large plasmid

[Kaiping YAN]

clive tregaskes wrote:
> 
> 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

Dear Tregaskes:

Thank you for your kind assistance. I will follow your advice and try.
In fact, the insert which is subcloned into pBluescript is a mouse
genomic DNA. I want to isolate the plamid for targeting construct.

Best regards

Kaiping YAN