PCR triplet

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Tue Apr 19 08:28:08 EST 1994


I have encountered a similar phenomenon.  When I ran the products on an
alkaline gel, they disappeared, so I concluded that they were PCR
artifacts and I did not pursue it further.

> 
>    I do PCR on plaques directly to search sequence variants. The expected DNA
> product is 310 bp. On an agarose gel I started noticing a second fragment
> that migrates faster (by about 30-40 bp). This extra band is usually quite
> faint. Checking with an polyacrylamide gel I now see THREE bands. One
> around 1 kb, the expected 310 bp fragment and the faster migrating
> fragment. Very consistent; if there are PCR products, there are always
> three bands. The bands are very distinct, i.e., no background smear. Since I
> plan to try cycle sequencing with the PCR products directly, I need help
> figuring out what's going on. I don't want to start off on the wrong foot.
>    Since I'm searching for additional clones of something I've already
> sequenced, I'm certain there are no other priming sites 40 or so bp away.
> Could the faster migrating fragment be single-stranded DNA? Since both
> primers should be at a final conc of 0.4 uM, it's hard for me to imagine
> that one of the primers could be in such limited supply. Any suggestions
> would be greatly appreciated.
> 
> Shiao Wang
> University of Southern Mississippi
>  
> 

==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
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