Tm of PCR primers: one 68, the other 58. Impossible to succeed? - (Dec/29/2008 )
Because of some reasons, my two primers have distinct Tm! I've noticed this problem before I did PCR, but I had to try.
I've tried twice --- no desired amplification, but I saw a light band with and without template (amplification of primers?).
first time: 94C - 58C - 72C
second time: 94C - 54C - 72C
(Note: in the first time, I was also doing some other PCRs, I set the 58C for them. These amplifications were OK.)
Anyone has suggestions? Or I should order another primer that has similar Tm (but no other choice)?
THX!
Only the part of your primer that binds to the template is used to calculate the tm. If the primer is 100bp long and only the last 20bp binds to the template, the tm is calculated from that 20bp.
However if the part of the primer bound to the template really has a tm of 68C and tm 58C, I think you might be out of luck. Buying a new primer would be the better option.
However if you want to try, you can reduce your annealing time to 5sec. Use an annealing temperature of 54C. See if that help. It probably won't. But try it interest sake. Dropping the annealing time has work for me when I was using primers (both primers) with an annealing tm of 65 and the lab had run out of betaine.
Thank you! I guess I will give it a last shot
Life would be much easier if started right.... 
However if the part of the primer bound to the template really has a tm of 68C and tm 58C, I think you might be out of luck. Buying a new primer would be the better option.
However if you want to try, you can reduce your annealing time to 5sec. Use an annealing temperature of 54C. See if that help. It probably won't. But try it interest sake. Dropping the annealing time has work for me when I was using primers (both primers) with an annealing tm of 65 and the lab had run out of betaine.
Aint' hard to redesign a new primer perhaps eliminate the 68 and get one closer to 58.