Robert Horton 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

More information about the Methods mailing list