mysterious PCR question

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue Jul 17 17:28:37 EST 2001


James,
	Sometimes I get a result like this with too much template.  I've
usually rationalized this as having a lot of template early that will
self-anneal out of register at low probability and the be amplified.
	Anyway, when I am reamplifying I do a set of serial 10-fold dilutions
of template and usually get one or two reactions that have the desired
band.  Your mileage may vary.

Warren Gallin

"Dr. James J. Campanella" wrote:
> 
> Hi everyone,
> 
> I've got a quick PCR question. I just amplified a cDNA of a
> gene of interest using RT-PCR. I gel purified the cDNA and
> isolated it using Gibco-BRL's gel DNA extraction kit.
> There was not much cDNA from the RT-PCR so I took the
> purified DNA and amplified it by an additional PCR rxn
> using primers known to amplify the sequence.
> 
> When I ran it out on a gel, I got a huge amount of DNA
> from the rxn, but it was a high molecular weight smear
> from about where the cDNA would have been at about 1400 bp
> up to perhaps 8000 bp. Yes, a very big smear.




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