Do not CIP your vector, and normal oligos work well. here is a link someone discussed before.

https://www.researchgate.net/post/shRNA_annealing_and_cloning

I suppose that the annealed fragment that you are trying to clone has the required sticky ends fitting the cut XhoiI and AgeI sites. No need to treat either the vector or the fragment with PNK (the restriction enzymes will leave 5' phosphates at either end of the vector). However, did you check the molarity of the fragment to be inserted ? Since it is presumably very short compared to the vector, it is easy to add a whopping molar excess relative to the vector. This will jeopardize the circularization of the plasmid: once a non-phospharylated fragment gets ligated to either end of the vector, ligase cann no longer close the construct

Digesting your vector with restriction enzymes will always produce phosphorylated ends, so no need for PNK treatment on the vector.

Theoretically, only one of the fragments (vector or insert) need to be phosphorylated to allow ligation. You should therefore be getting colonies. Did you verify proper annealing of the oligos on a gel ? Are you sure that your competent cells are good ? Finally, did you purify your digested vector prior to ligation ? If your small Xho-AgeI digested fagments are still in solution, they can definitely bind to your insert and prevent it from binding to the actual vector.

If you have not treated your digested vector with a phosphatase (like alkaline phosphatase) then your vector has the required P group for ligation. If insert doesn't have a P group at the ends it is not a big deal in this case cause ligase can make the bond and subsequently repair the produced nick.

About the self ligation: with these enzymes producing sticky ends if you give enough time for enzymes to cut both sites so it is acceptable not to have grown colonies.

Overall, I guess it could be better to check the other factors having impact on ligation especially vector:insert ratio, your buffer etc.

Junjie Shao Saber Delpasand Khabbazi Thank you so much for the inputs.

Martin Chenal Yes I have purified the digested vector prior to ligation and checked on the gel as well, a single sharp band of the desired size. And do you think I can see the annealed oligo? The size is around 50 bp. Even if I run a 2 or 3% gel, would I notice any difference between non-annealed and annealed oligo?

Pierre Béguin Professor, thank you so much for the explanation. Well, about the molarity, I checked the concentration of both vector and insert prior to putting ligation and used the 1:3 vector to insert molar ratio for the ligation.

I have started everything today from scratch again. Will try and will hope for the best.