Overlap/Fusion PCR smears

[cat1082]

Hi everyone,

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?


Thanks,
Cathy

[David Ling]

I have the same problem, waiting for expert!

[jonoave]

This link was helpful to me when running overlap PCR:
[Only registered users see links. ]

1. I think the critical step is the initial annealing and extension of the fusion template. Try running a gradient starting from 50C (e.g.+1).

2. The amount of PCR products you use as a template could have an effect as well. If you keep getting smears, try diluting both the PCR products you use.

[Lina Sun]

Hi, guys, I read some references about it, less than 5Kb fragment was got, wasn't it? I want to know how long fragment will be get in this method? Could 15Kb?