Isolation of plasmid DNA from transfected eukaryotic cells

Nico Dantuma nico.dantuma at mtc.ki.se
Sat Sep 5 09:37:53 EST 1998


Hi,

Isolation of episomal plasmid DNA from transient transfected cells is one
of the steps used in expression cloning of genes. I haven't done this
myself but I propose to check the literature on expression cloning. In
expression cloning episomal DNA is purified from COS cells that are
transient transfected and selected for a certain phenotype. The isolated
plasmids (which is because of the selection part of the initial library) is
propagated in bacteria, purified again and used for another round of
transient transfection of COS cells + selection. I don't know anything
about the quantities or purity.

Good luck,

Nico


>Resent-From: server-daemon at dl.ac.uk
>Date: Fri, 04 Sep 1998 16:41:31 -0700
>Resent-Date: Sat, 5 Sep 98 0:0:52 UT
>From: bpmurray*STUFFER*@socrates.cgl.ucsf.EDU (Bernard P. Murray, PhD)
>Reply-To: bpmurray*STUFFER*@socrates.cgl.ucsf.EDU (Bernard P. Murray, PhD)
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>To: "bionet.molbio.methds-reagnts mail newsgroup" <bionet-news at dl.ac.uk>
>Subject: Re: Isolation of plasmid DNA from transfected eukaryotic cells
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>In article <01bdd7c9$b4b808c0$1aafd8cc at default>, "Gonzales"
><hojimo at cts.com> wrote:
>
>> Hello All!
>> 
>> I'd like some advice on which protocol to use to isolate episomal plasmid
>> DNA from mammalian cells which have been transfected via CaPO4
>> precipitation. Is there a method that will yield clean plasmid with a
>> minimum of chromosomal contamination? Is such a thing even possible? Or
>> does a good genomic DNA prep have enough plasmid in it to use in southerns
>> and PCR experiments?
>> Along the same lines, I'd like to know if anyone has used a good DNA
>> isolation protocol and PCR to detect either episomal or integrated plasmid
>> in tissues of animals. 
>> Thanks!
>> Jose E. N. Gonzales
>
>As long as you don't need too marvellous a recovery you could try
>and extract the plasmid from cellular cytosol (I assume there'll be
>some plasmid there) using methods akin to RNA extraction.  Basically
>you gently lyse the cells with detergent (eg. cholate/NP40) and then
>spin down the nucleii with the cellular debris.  The supernatant
>should have little chromosomal DNA but good levels of RNA and
>(hopefully) plasmid.  Then do a phenol extraction as you would for
>RNA but substitute buffered phenol for the "acid" phenol normally
>used.
>     Maybe there's some literature reports on extraction of SV40 or
>EBV based vectors (which can be propagated episomally).
>
>I am not sure what you mean in your "along the same lines"
>question.  If you simply want to extract genomic DNA for
>PCR there are some nice methods based on proteinase K digestion
>(I don't have my favourite reference to hand).  However, if you
>want to distinguish between integrated and episomal DNA that would
>be a little more difficult - maybe something like inverse PCR
>would work, especially if your episomal DNA is circular.
>     I hope that this helps,
>          Bernard
>-- 
>Bernard P. Murray, PhD
>Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
>
>
>
------------------------------------------
Nico Dantuma
Microbiology and Tumor Biology Center
Karolinska Institute
Box 280
S-17177 Stockholm
Sweden
Phone: + 46 8 7286281
Fax: +46 8 331399 	



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