In PCR, we need to determine the optimal annealing temperature, which is ~5C lower than Tm. I am running RACE using SMARTer RACE kit which require primers with Tm of >70C. I have a set of primer design using primer 3. The calculated Tm is 76C (Tm=4(G+C)+2(A+T)). However when i buy it the label on the primer says that the Tm is 68C. That manufacturer use another model to calculate Tm.
Now, generally Ta is around 5C lower than Tm. Which Tm should i use? I've tried Ta of 65C and 68 using normal pcr and it had terrible smear. Should i increase or decrease the Ta? I am aware that doing gradient is the best solution but i don't have a gradient cycler and i have 10 samples using different set of primers. Any tips to predict the range of Ta?
Edited by hianghao, 07 October 2010 - 06:06 AM.