I want to delete 45 bp immediately next to the signal peptide in my gene of interest. I have designed 2 primers at either end of the the segment that I want to delete in such a way that the whole plasmid pUC18 is amplified except the segment. I purified the PCR product, digested DpnI, loaded and extracted from the gel and eluted in elution buffer provided in the kit. I set up ligation in a 20ul reaction. I have to religate my PCR product which is a whole plasmid. I used 5U of T4 DNA ligase, 2 ul of buffer and 2 ul of PEG 4000 with nearly 100 ng of DNA, and incubated at RT for 3 hours as well as overnight.
I did transformation but I see no colonies on the plate. I tried the ligation condition without PEG as well. I tried a couple of times using different comp cells. My positive control with the original plasmid worked.
Can you suggest anything else I could try or where I might have made a mistake?
Are you using phosphorylated primers or phosphorylating the PCR product after amplification? You can not ligate DNA that is missing the terminal phosphates at both ends.
Thankyou Prof Michael Benedik. I have not phosphorylated my PCR product but Im not sure of the primers being phosphorylated ( I don't think they are) . I will do the phosphorylation of my product and perform the ligation again.
yes, that should work. The other option would be to create different primers that generate the deletion you want in a single step where you don't need to ligate.
Sachin, that is NOT correct. Primers are NEVER with a 5' phosphate unless the phosphate is specifically added, remember they are made synthetically and not biologically.
Thanks a lot for the suggestions.. I will try using T4 PNK. If not I will order new primers with phosphorylated ends... I think this should solve my problem :)
Probably the phosphorylation of the primers is your main problem, but keep in mind that almost every Taq polymerase, except the Pfu, is adding at least one extra adenosine at the 3' end of a variable percentage of the amplified strands, so unless you took specific care of using the Pfu for your PCR amplification you'll have, depending on the Taq you used, a more or less large fraction of your amplified fragments with an A overhang that will make a blunt ligation impossible. If after the phosphorylation step you should still have problems ligating your vector you should therefore first blunt your PCR fragments, easiest with Mung Bean exonuclease.
Dear Pietro Pilo Boyl,
Thankyou for the suggestion.Im using Phusion polymerase for the PCR
That's good, the Phusion adds the A in about 50% of the amplified fragments, so you shouldn't have too many problems with the blunt ligation if you phosphorylate your primers. Good luck!
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