I have tried amplifying my individual fragments using Phusion but it is not working consistently but always works fantastically with KAPA. I think I am unnecessarily wasting time on Phusion. Thinking of switching to KAPA pol, not just for this individual fragment extension but also for overlap PCR. has anybody tried it?
Hi, I think Fusion is a good option and is similar to KAPA for overlap PCR. But my recommendation is that you can used inclusive Pfu and use primers separated by 12 cycles and then you mix each one in a just reaction and then you put this in the PCR conditions convenients for 30 cycles, you could add more polymerase or not. I hope help you
I am assuming that you have optimized conditions for amplification for each enzyme individually. If not, you may consider doing it.
I would recommend pfu polymerase; I have never encountered any error in sequence that was introduced during amplification.
Best,
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