I read protocols partitioned into two parts: the first performed an annealing temperature gradient, and the second used a single annealing temperature. My question is about the parameters that I should consider for the second annealing temperature.
Frederic Lepretre
Hi Isis
long time I've done one
it's easy, let say that your Tm is 60°c, after the denaturation cycle you will make 20 cycles beginning at 70 and decreasing by 0.5°c at each cycle, and finishing with 20 cycles at 60°c.
you can use short times and 5% DMSO to enhance reactions.
best
fred
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