I am trying to fuse a 500bp fragment to a 2500bp fragment with overlap or fusion PCR, whichever term you prefer. I haven't had any success so far as I've been getting either blank gels or smears.
I have been using very rough estimates of the right amount as I've been too lazy to properly quantify my separate PCR products to be frank, but I'm not sure what sort of ng amounts I should be using. I'm aiming at roughly equimolar amounts none-the-less.
I run 10 cycles with everything added barring the forward and reverse primers at a rather low annealing temperature (50 C) with Phusion for 3 mins extension time. Then I add a smidge more enzyme and the primers (10pmol/ul stock concentration) and run for 35 more cycles which produced a smear, or 25 cycles with produced nothing. The conditions are the same i.e. 50 C and 3 mins.
If anyone could suggest any improvements or give me a better protocol I'd hugely appreciate it. I read somewhere you should use a higher annealing temperature for the initial 10 cycles?