how to isolate a large plasmid
agriff at aa.net
Thu Jun 10 17:57:58 EST 1999
Kaiping YAN wrote:
> 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
I used to have to isolate an 85 kb plasmid from E.coli (copy number 1).
I did a standard Alkaline lysis prep, but what made a big difference was
turning the acceleration of the centrifuge down when spinning out the
protein and genomic DNA. If you don't have a variable speed centrifuge,
you can spin them at low speed for 5 minutes (2000 x g or so) first,
then turn the speed up. I think that all the precipitating goo tended
to trap the larger plasmids when it got spun out to fast.
The Qiagen plasimd kits also worked pretty well, with the above spin
speed modification and double normal solution volumes.
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