Usually cloning like this results is way less colonies. You should do your ligation with PEG4000 in the reaction and double your ligase concentration, I would use 1:1 ratio for all fragments involved. Having said that you do not have to throw away your old ligation reaction you could amplify from your previous ligation using primers that amplify the entire ligated fragment( so forward primer you used for insert1 and reverse primer for insert3) then digest that amplicon with aseI/kpnI then ligate to your vector (digested aseI/knpI), which would make it a single fragment ligation which is way easier.

I agree with Hanna Alalam. Additionally, sometimes the size of the final product after inserting all the fragments into the vector tend to be too large that its transformation into competent cells become problematic. You may try to increase the transformation incubation time but not too long that will affect the cells.

Again, the you should also check the concentration of the antibiotics used for selection. If it is too high it could also lead to no result.

Another possibility is the strength of the promoter used. Is it a native promoter? Is inducible? Probably the recombinant DNA was successfully transformed but its selection marker gene is not expressed. Check all possibilities.

1. how about the competent cell efficiency?

2, yes as Hanna said, you could do fusion PCR first to get a fusion fragment.

Thank you all for your answers! Just a little bit more information on what I tried already: 1) yes, I tried to ligate 3 fragments and to amplify the entire thing, did not get any PCR product 2) tried to ligate fragments in pairs (1+2 and 2+3). That gave me a product and I tried to subclone it into pJet. That only gave me colonies for 1+2. Thanks for all of your advice again. I will try to check cells and I will use your advice of ligation mixture. will see how it goes!

1. What is the commercial target vector? What is the maximum length the can be inserted? As previously mentioned, some vectors become unstable if too big.

2. Do you have fragment #3 in Pjet? Can you cut it out?

3. Try to cut out 1+2 from PJet, #3 from Pjet and ligate to cut vector.

4. Make sure you use the correct antibiotics.

Note: While cutting PCR products and ligating them may work fine, sometimes one of the restriction enzymes may not work and you won't be able to see it. It is always recommended to ligate fragments cut out from sequenced plasmids. The "long" way is usually the shortest way.

If I were you, I will do step by step, clone the 1st insert into the vector then the 2nd insert , last with the 3rd insert, or doing overlapping PCR to fuse 3 insert in 1 first, then you can clone as normal.

You can try this way: Put fragment 1, 2 and 3 in one vial, use fragment1 forward and fragment 3 reverse primers, amplify the full-length fragment 1, 2 and 3, then purified the fragment and ligate to your vector. If it not work, just add more than 18 bps overlapping between fragment 1 and 2, 2 and 3, good luck!

Also, you can try assembly cloning strategy, it is more convenient than traditional enzyme ligation strategy

For a PCR cloning project I would make sure your RE sites are present and are cutting effectively. As suggested by Gloria you have no real way of knowing if RE are effective just by gel analysis: you could always sequence the PCR? Clone fragments into vectors and sequence individually. This way you have infinite supply of fragments to trouble-shoot ligations.

I may also consider overlapping PCR, so re-do PCR and incorporate at least 18 bp overhangs on each fragment as suggested by Aiguo Xu.