IUBio

PCR

Rich Dudley rdudley+ at pitt.edu
Tue Mar 2 08:52:35 EST 1999


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.
>
> Thanks
>
> 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.

Good luck!

rich

--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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