For TD PCR, it's recommended that the annealing temperature (T_a) be 10°C higher than the normal cycling T_a for the first 10 cycles or so, dropping 0.5 to 1.0°C/cycle. Should the extension temperature (T_ext) match this pattern to prevent miss-annealing? It seems that if the initial T_a was 75°C, then T_ext was set to 72°C (or whatever is optimal for the polymerase), the purpose of the high T_a would be lost.

Instead, should T_ext match T_a with an increased extension time until the optimal T_ext is reached?

  • $\begingroup$ Your initial annealing temperature 75°C would mean nomal cycling annealing temperature 65°C. That is really high. Is there a reason why you did not design your primers with Tm ~60°C (normal annealing ~55°C)? $\endgroup$ – BagiM Oct 17 '19 at 6:18

I do not if I understand correct the question. But I write you an example. For example for the amplification of the gene nirS, I used a touchdown PCR. The Tm of the primer is 60°C. I created a touchdown from 65°C and each cycle decrease of one degree (total cycle of the touchdown is 5). The thermoprofile is the following: 94°C for 3 min; 5 cycles of 94°C for 15 sec, 65°C (touchdown -1°C) for 30 sec, 72°C for 30 sec; 34 cycles of 94°C for 15 sec, 60°C for 30 sec, 72°C for 30 sec; followed for 72°C for 10 min. Is better that in total your PCR do not make more than 40 cycles. I hope that this help you Jo

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