It looks like none of the forms visible in the gel patterns corresponds to the circular form of the recombinant plasmid you are trying to obtain. One possible explanation could be that one of the restriction enzymes that you use to treat the insert in order to generate the sticky ends required for ligation into the vector didn't work, so that circularization does not occur. This possibility is also supported by the absence of concatemers in the gel patterns.

Thank you for your answer ! Concerning the restriction enzyme efficiency, I tried to digest the same plasmid contaning a previously cloned insert, with the same restrictions enzymes (NdeI/XhoI) and I obtained 2 bands corresponding to the insert and the linear empty plasmid on the gel.

Also, are you sure that none of the forms correspond to my circular plasmid ? Because in term of size, the upper band could be the correct one (but it's hard to see with such intense markers, I should try with a lower quantity). It does not appear as a smear but as it has been previously digest with restriction enzymes and not cloned into bacteria (i.e it has not been in the presence of any topoisomerase), I think it should not be supercoiled, thus not appearing as a smear.

Assuming you're adding restriction sites on the PCR primers for your insert, are there extra bases before the restriction site? Some restriction enzymes will have trouble cutting near the very ends of linear fragments. There's a chart from NEB about this: https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

Both NdeI and XhoI require some extra bases for efficient cutting.

Thank you for your help :) Yes I already added some additional bases depending on the optimum for each enzyme. At this point I am checking with individual restriction (NdeI alone and XhoI alone) and then ligation if there is an issue, by any chance, with one of the end of the primers.

How are you screening for insertion? Does your insert confer any kind of antibiotic resistance, or are you using blue-white screening?

I'm not familiar with using NdeI / XhoI as my restriction enzymes, but if you think that some of your plasmid has circularised, you may need to desphosphorylate the plasmid before adding the insert to prevent it recircularising and excluding your insert.

What is your positive control? Is it your plasmid with no insert added, ligated?

Thank you for your help again, and sorry for the late response, I was trying new ways to succeed these transformations. My insert doesn't confer any antibiotic resistance, but my plasmid does, and because of the double digestion I supposed that it will not self-ligate. My positive control is the plasmid ligated with an insert generated from PCR, and this insert has been previously successfully ligated from previous experiments. By the way, I don't think that there is any self ligation or similar issues, because I don't see any colonies, even for my supposed positive control.

I am currently trying to clone with another method (NEB HiFi DNA Assembly), and I think that if this work, I will use it for my other constructs, but all of these issues with the classical cloning method are quite disappointing. I discussed with experimented colleagues, check all of the steps (for example I tried ligation with fresh buffer and without agitation recently, but this didn't worked), and I don't see any thing that could explain my issues.

By the way, thank you again for your help !

What are you using to stain and visualize your DNA? If you have the materials to do so, I highly recommend something like sybr green rather than ethidium bromide. The time that it takes you to find your bands and cut them out is enough to damage your DNA, making viable ligation difficult.