PCR of episomal plasmids
David L. Haviland, Ph.D.
HAVILAND at KIDS.WUSTL.EDU
Fri Jun 3 23:45:45 EST 1994
In <1994Jun2.175239.24391 at emba.uvm.edu> brianf at med.uvm.edu writes:
> Rosemary Lemons (rmlemons at um) wrote:
> : Any hints on how to PCR plasmids that have been transfected into
> : fibroblasts?
> : We know they're in there but have yet to obtain any PCR products. I can
> : get
> : product using just a plasmid at the femptagram level so I believe I've
> : optimized conditions, but still nothing from the cells. Any thoughts would
> : be
> : greatly appreciated as we're a bit new to this area.
> How do you isolate the template DNA? If the plasmids are still
> episomal, not yet integrated into the genomic DNA, then they may not
> preciptitate eficiently with the chromosomal DNA.
> Have you tried PCR from whole cells? This never worked for me, but
> I did not try too hard. There are numerous protocols for using whole
> cells in PCR.
Brian's suggestions are quite good. I'd like to add my $0.02 worth - I'd
also consider the Hirt method which in your case should specifically grab
the episomal DNA at the expense of the chromosomal. (Hirt, B (1967) J. Mol
Biol. 26:365-369) I do it exactly as Hirt describes but extract (clean up)
the resulting supernatant with 2 phenol, 2 phenol/clfm, and 2 clfm
extractions prior to EtOH PPTing down the episomal DNA. I've taken this
method as far as transfecting COS cells with a plasmid (control w/o
plasmid), let it go for 72 hours as it is a transient transfection, "Hirt"
the cells, PPT the DNA and transform a small quantity into bacteria (Sure
cells). At that time I determined success by obtaining transformants but I
did not take the transformants any further than that - i.e., mini preps
with subsequent digestion and sequencing to see if it was what I started
with... however, I doubt that something other than what I put into the
cells would spontaneously confer ampicillin resistance! BTW - the negative
control resulted in a blank plate. In short, I don't see where this
method wouldn't yield episomal (partially pure at that) DNA that you could
use for your PCR studies.
Hope this helps,
+ David L. Haviland, Ph.D. Internet:"haviland at kids.wustl.edu" +
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