PCR triplet

Shiao Y. Wang sywang at whale.st.usm.edu
Mon Apr 18 22:56:47 EST 1994


   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
 



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