The primers i've designed have 5' overhang, which will be used for gene in/del (based on homologous recombination in yeast). To amplify this fragment, the annealing temperature of the first rounds must be lower than their annealing temperature, right? So, how low it must be?
Any suggestion will be very welcome!
Hello
Usually you use the annealing temperature for the non-tail region for the entire amplification. So use the annealing temperature of the region that actually binds to your template (the actual temp you use depends on which polymerase you use). for argument sake let's say you are using taq pol which requires an anneal 2-3 less than the calculated temperature of the template binding region. So. Let say the anneal region tm(without the overhang) is 57 when using taq pol run the entire amplification at 55 (even if the to of the entire primer is actually higher than 57 when you calculate including the overhang). It is unnecessary to change the annealing temp in later cycles. I attached a small image to help you visualize it.
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