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')