It is not clear what you mean by compatible sticky ends. If each fragment has different ends so that there is only one way to assemble, then you can just add roughly equimolar amounts of the three fragments and ligate like normal. I would do control ligations with the various pairs just to know what the background is.

On the other hand if all the fragments end with the same sticky end, then there are numerous combinations that could arise. This becomes much trickier because there are many possible outcomes.

dear Dr. Michael J. Benedik

thank you so much for your comment.

actually, my fragments are regions of my vector that I digested, after digest my vector was cut into 3 fragments (2400,1500 and 200 bp), any 2 ends of the fragments are compatible with each other only, as you "each fragment has different ends".

do you suggest me to use polyethylene glycol?

in T4 ligase ،hermo Fisher noted time and temp of ligation should be 22 centigrade for 1 hour. Are you ok with this time and temp?

22c for an hour should be fine. Whether PEG would help depends on the DNA concentration. If you have plenty Of DNA then its not needed but won’t hurt.

the problem you will have is that there may be a dozen or so different configurations that you will need to screen through. Call your 3 fragments 1, 2 and 3. If the origin and resistance gene are on 1, hen you could get 1 only. 1+2, 1+2reverse, 1+3, 1+3reverse, then 1+2+3 in all combinations of forward and reverse. Added to that you could have 2 copies of either 2 or 3 in all of the above.

It’s cleaner if both 1+2 are needed for replication and resistance, otherwise you are likely to mostly get 1 alone.

Dear Dr. Michael J. Benedik

I am thank you so much

you really help me.