So I have tried to PCR a fragment for my assemblies. (not so successful as of late I might add) although I have done them almost 100 times.

I have changed polymerases (not related to my unsuccessful assemblies) (went from Q5 2X MM to HighQU Allin Mega HighFI MM).

What do you think may have caused this? You can see I also have some non specific amplification.

1) Primer anneal temp was 57 C (used the same anual with Q5 and worked well). In the HighQU protocol it is said that anneal should be 60-65)

2) Something wrong with my template?

3) I have added a 72 C extension for 2 minutes at the end of the 30 cycles.