purification of 10 to 100 mg of plasmid?

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Thu Dec 10 16:49:42 EST 1998


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
> > : HALLO NETTERS:
> > : Please, I am looking for a IN HOUSE protocolle of
> > : plasmid purification in order to get 10 to 100 mg of
> > : plasmid for inmunization whith DNA,

> > Why don't you just scale up the Merlin prep.  1 gm of resin can
> > binds up to 2-3 gm of DNA,

Is this a typo?  Shouldn't that be 2 - 3 milligrams of DNA?

> >therefore making yourself a column of
> > this resin should be enough for very large scale prep.
> > See
> > http://www.public.iastate.edu/~ippm/merlin_miniprep.html
> > and
> > http://www.nwfsc.noaa.gov/protocols/diatom-maxi.html

> 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



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