PCR of episomal plasmids

Brian Foley brianf at med.uvm.edu
Thu Jun 2 12:52:39 EST 1994

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 Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *

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