Setting the temperature range for gradient PCR - (Jun/09/2014 )
I was just wondering what is the protocol for determining the temperature range when running gradient PCR?
I don't know the calculated annealing T but I do know the Tm of the primers. If the Tm of the primer is 56.6 degC, what should be the temperature gradient?
For normal GC ration organisms, I would use a range of 48 - 66 degrees. For high GC organisms, you might want to raise the upper limit to 70-72 (or do a two-step anneal/extend).
I have been using 54-62 degC. Would this have caused any issues?
That range shouldn't cause issues, but you might miss an optimum at beyond either end of the range. What I would do is use the range you have, if that doesn't work, try a new range up to the Tm but overlapping with the range you have already used, and then if that doesn't work either, go for a lower range.