RF-Cloning.org Forum

Hi Steve/all,

I am attempting RF cloning and ordered primers from a paper that are completely complementary, the 5' and 3' ends both match the target plasmid (32nts on each end). The insert is 30nts, and is replacing 15nts in the recipient plasmid, so in total the primers are 94nts long.

When I used this website to design the primers, they were not complementary on both ends, should I anneal these two together? I've tried the cycling as in the paper several times- and after dpn1 digestion I keep getting the original recipient plasmid.

I've tried adding the first primer + plasmid, running five cycles and adding the second primer then 30 cycles- and as written in the protocol, neither worked.

Primer1: AGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCGCTCTTCC-Insert-GCTCTTCTGTCAGCCATGGGGCGGAGAATGGGCGGAACTG

Primer2: CAGTTCCGCCCATTCTCCGCCCCATGGCTGACAGAAGAGC-Insert-GGAAGAGCGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCT

Thank you!