I love Gibson assembly. It works almost everytime
(can’t help but put this here).
CPEC works by creating amplicons with overlapping 5′ and 3′ ends that are complementary to the 3′ and 5′ ends of neighboring DNA fragment(s). The complementary ends have to be long enough to generate an annealing temperature greater than or equal to 60oC. For a CPEC PCR, you assemble as PCR with your high-fidelity polymerase as usual. Instead of adding primers and template to amplify, you add the fragments with end-complementarity. In my hands 55oC annealing works nicely. Then I perform extensions that are sufficiently long to circle the entire circularized vector; this works out to about 15 seconds / kilobase for Q5 polymerase. At least 12 cycles are required. That’s it. Transform the product or examine it on a gel. You’ll see a smear below and above a band corresponding to your fully circularized vector. I was worried that using a polymerase would cause amplification errors. But there are no primers with which to create amplification errors. Again, in my hands I have not observed errors as a result of CPEC.