RF-Cloning.org Forum

Hi Sand,
You're not alone in misunderstanding how the rf-cloning method leads to a circularized product. Some mental gymnastics are involved.
It all has to do with the positioning of priming sites. Both the 3' and 5' end of a good mega-primer will be bound to it's target parental plasmid, with some non-complementary sequence in the middle. As you know, priming is initiated at the 3' end, and then, because the DNA polymerase does not have 5'-3' exonuclease activity, synthesis stops when the daughter strand bumps into the 5' end of the mega-primer. This happens on the complement strand as well, and two nicks are present when these newly synthesized products anneal — offset by the length of the mega-primer.
In the next round of PCR, if a mega-primer binds to a daughter product, the 3' end it finds itself at the extreme 5' of the template. Meaning there is nothing to synthesize in the 5'-3' direction, and this is why the 2° PCR of an RF-cloning project is non-exponential.
I hope this clarifies, but I can imagine it's still a bit confusing. The best I can do is promise that the logic is sound, and encourage you to draw the process out on paper until you see how the pieces all fit together. You've probably already looked at it, but it might be useful to check out the diagram in the Q & A again.
Take care,
-Steve