purification of 10 to 100 mg of plasmid?

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Thu Dec 10 21:37:06 EST 1998


In article <bpmurray*STUFFER*-1012981449420001 at macmac-2.ucsf.edu>,
  bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) wrote:
> In article <74pcuj$1gb$1 at nnrp1.dejanews.com>,
> theodorn at medlib.georgetown.edu wrote:
>
> > In article <1998Dec10.161639.37231 at ucl.ac.uk>,
> >   chen at bsm.bioc.ucl.ac.uk (Chen Ho An) wrote:
> > > JUAN MORALES GRILLO (Juan.Morales at CIGB.EDU.CU) wrote:
> > > : To:                 methods at net.bio.net
>
> > One thing that Juan might want to keep in mind is that for immunizations the
> > DNA should be endotoxin free. I was under the impression (and someone please
> > correct me if I'm wrong) that ion exchange resins are better at removing
> > endotoxin from DNA than silica.
> > Nick Theodorakis
>
> I have the impression (albeit not tested formally) that the
> endotoxin binding property is of true silica (eg. Promega
> Wizard) rather than diatomaceous earth (eg. Promega Magic,
> "Merlin").  Certainly the diatomaceous earth doesn't have
> the problem that silica has in binding endonuclease and
> binds RNA less than silica.  You can still remove
> endotoxin from solutions with binding agents (I think Sigma
> sells such a kit).  There are some simple endotoxin assay
> kits available (Sigma again) to check your samples.
>      An alternative would be a boiling maxiprep followed
> by PEG precipitation.  The DNA is probably less clean but
> its good enough for cell transfection.
>      Good luck,
>           Bernard
> --
> Bernard P. Murray, PhD
> Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

Well, I had heard it about true silica, too, and just assumed it was also
true about the diatomaceous earth, because aren't diatom "skeletons" made of
silica? I thought that the differences between the two were due to the higher
surface area of the diatoms, which intuitively might lead one to expect it to
bind more endotoxin and stuff.

Nick Theodorakis

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