I have done many of these types of PCR and have extensively tested various Tms
for various lengths of matched/unmatched/extended regions.
I can tell you that the polymerase treats the primers as though the unmatched
extended portion is nonexistent!!! I once had a primer with a match of 18 bases
and an extension of ~50 bases and the PCR reaction worked at the temperature
predicted for the 18 bases.
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')
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