mysterious PCR question

Dr. James J. Campanella campanellj at mail.montclair.edu
Tue Jul 17 15:34:04 EST 2001


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. 

Any idea what would have caused this? Using specific
primers I should have gotten a single band?! I checked the 
gel purified cDNA by electrophoresing an aliquot, but it was
clean with no contaminating DNA.

Thanks,

Jim
----------

James Campanella,
professor
Montclair State University
Dept. of Biology
1 Normal Avenue
Montclair, NJ 07043


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