genomic DNA in plasmid prep and Plasmid Safe

Philip Carl plc at med.unc.edu
Tue Jul 28 15:01:47 EST 1998


fwhyde at ibm.net wrote:
> 
> John Dixon wrote:
> 
> > > This may not work on Sundays or Tuesdays or whenever your time is
> > most
> > > pressed. :) I've yet to find any way of ridding samples of
> > chromosomal
> > > once its in the prep. (Tried some acid phenol, which can work if the
> >
> > > plasmid is not also removed.)
> > >
> > > If you can solve this, you will be a hero. (Plese E-mail me if you
> > have
> > > a clue).
> >
> > Has anyone tried the PLasmid-safe enzyme from Epicentre I think? I got
> > a
> > flyer about it once. They have some sort of exonuclease that digests
> > linear dsDNA and closed circular ssDNA and linear ssDNA, but can't
> > touch
> > nicked/closed-circular/supercoiled DNA.
> 
> Epicentre's Plasmid-Safe product number E3101K (1000 U), E3105K (5000
> U), E3110K (!0,000 U).

I did try Plasmid-Safe on two occasions and was unable to see any
digestion of chromosomal DNA using an ATP solution and the buffers
supplied with the enzyme.  I certainly would not claim on that basis
that the enzyme does not work as claimed, only that I was unable to get
it to work.  I have been meaning to contact Epicentre to see what they
can suggest, but haven't gotten around to it.  In the meantime, I hope
others will contribute to this discussion, as in principle this enzyme
is a very valuable reagent, and should be very useful if it is as easy
to use as is claimed.

The only other observation I could make is that various plasmid prep
protocols differ a good deal in the way they spin down the precipitate
of KDS, protein, and chromosomal DNA.  Qiagen uses a 30 spin, but many
other protocols call for far less.  I suspect that this step may be very
important in removing the traces of chromosomal DNA that can contaminate
plasmid preps.
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