horton at molbio
Fri Jun 5 15:20:53 EST 1992
URA1335 at FRCITI51.BITNET writes:
: Hi! Netters,
: I have a PCR problem!
: According to Tm=2AT+4CG, we calculated a Tm=66
: for oligo 1, and tm=58 for oligo 2.
: Here is my question : how come that i had still
: PCR bands ( that I have no any idea about their
: specificity ) when i did my PCR reaction
: at an annealing temperature = 60 degrees ????
: just 2 degrees above Tm.
Tm is just a guess at optimal annealing temperature. Primers seem to
work at least 5oC above Tm, or more. Its an excellent approximation
of the melting temperature of an oligo when used as a hybridization
probe in 5XSSPE, tho.
: is it necessary to continue to do PCR in highter
: temperature above Tm ????.
Maybe, but if you're trying to increase specificiy, I'd suggest hot
start. It can make a phenomenal difference ( a well kept open secret).
Bob Horton /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS || with nine women pregnant you get a baby a month"
1479 Gortner Ave. /||\ -Werner von Braun.
St. Paul, MN 55108 ^^ horton at molbio.cbs.umn.edu/(612) 624-3790
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