Rick L wrote:
> Dear all,
>> We got a problem fpr PCR-cloning a gene. The primers we are using are very
> long(~60 bases), but for each primer, only 29 bases are complementary to the
> template(we have to use such long unmatched extensions). I wonder how to
> caculate Tm. On the other hand, I'm not sure if I can use a standard method
> (ie, 94C, 45s; Tm-5C, 30-60s; 72C, 60s; 25-30 cycles).
>> Your help will greatly appreciated.
>> Rick(change 'll' to '11')
Typically what I do is take the Tm of the matching regions, and run 5-10 cycles
at that annealing temp. Then I use the whole primer Tm, and run the next 20-25
cycles with that.
--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
---> search BIONET archives at http://www.bio.net <---