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
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