Hello,

I have successfully amplified 3 PCR products which contain overhang regions required for assembly PCR (around 25bp overlap). I tried running an assembly PCR cycle as follows:

1. From the previous amplified PCR product, I mixed the three fragments at a 1:1:1 ratio. I added primers that will amplify the entire product (The forward primer and the reverse primer of the two outer fragments)

NOTE: Is there a particular amount of DNA in ng that works better for this fragment mixing step?

2. I next ran a regular PCR cycle with an annealing temperature for the two outer primers.

I did not get any major amplification of the correct size. Anyone have ideas for how to perhaps modify my PCR cycle for the assembly process to work?

Thanks.

Adam