RF-Cloning.org Forum

jk1234

Member

Registered: 2020-08-27

Posts: 1

Ordered primers that make the entire insert - overlap on both ends

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!

Last edited by jk1234 (2020-08-27 20:18:35)

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Ordered primers that make the entire insert - overlap on both ends

Hi jk,
Yes, you should be mixing these two oligos together in equal molar ratio and move straight into the secondary PCR step. I believe I've found your project in the database but it looks like your megaprimer is actually 114 bp? Regardless, if the plasmid I'm seeing is correct (final size 7383 bps), you're looking at setting up a reaction with 27 ng total of your mixed primers and 88 ng of plasmid. Annealing temp 72C, extension time of 2:26 min, 15 cycles. What's the doi on the paper you've pulled these primers from? I'll have a look at their methods.
Give it another try and let me know how it turns out!
-Steve