I want to clone a 1300 PCR fragment into a 8000bp plasmid using NruI (blunt end) and HindIII (sticky), but I didn't get any colonies.
Here's what I did:
Being limited with potential sites, I am using NruI (no other choice) and HindIII. So I did PCR with restriction sites and 5' overhangs, which gave bands as expected. Then I digested my PCR product and vectors first with HindIII (Buffer NEB2), gel purified everything before second digest with NruI (Buffer NEB3). Ran that on gel and saw the bands of the fragments I cut out of the backbone as expected, gel-purified insert and backbone again before ligation. So far I think everything worked.
Then I performed ligation with standard T4 DNA ligase in a vector:insert molar ratio of 1:2, 100ng in total at 16C overnight. Used 2ul of ligation for transformation with TOP10 and plated plenty of it.
I think most likely the issue is the ligation, and I was wondering if there is any advice what to try first to improve it. Would you just try different molar ratios at the same time? Any particular issues for mixed sticky and blunt ligation? Would dephosphorylation help?
Many thanks for any help!!