I designed several primers for a directional TOPO-reaction.
But not all of them are working...Can you give me some tips how to determine
a good annealing temperature (except for gradient pcr)?
For example these to primers
a) CACCATGGAGATCCCGGTTCGA (22 bp TM = 53.4)
b ) AGTTGCTATTTTTTCTAATTTCTC (24 bp; TM = 46.8)
The TMs are calculated via Vector NTI. In case a) the leading CACC sequence is ignored
for the calculation because it is not really binding.
As a rough rule of thumb the annealing temperature lies 5°C below the TM. In this case it would
be 41.8 °C. A little bit low, isn't it?
Edited by Ikar, 23 April 2011 - 09:43 AM.