That's pretty difficult!!

Since you seem to already have the two fragments, I would suggest you to fuse them by PCR (Phusion Taq polymerase works pretty well for this) and clone the final product.

You can clone the product classically using the two RE A and C, or, since you design primers for PCR, you can design them for InPhusion or Gibson Assembly seamless cloning.

Firstly cut both the fragments with enzyme B and further ligate.  Now cut the ligated product and vector with enzyme A and C and ligate. Theoratically it seems like this only, practically it will challenging though. Good luck

What is the big problem in it ? Use 2 molar excess of both AB and BC digested  fragments and take AC digested vector and ligate at 16-22 temperature. You just need to keep in mind that digested and gel eluted fragments should be pure and T4 DNA ligase should be good along with good E. coli competent cells.

my problem now is these two fragments are separately in the different vectors, so if i want to get the purified one, i have to cut them out from the vector, so basically the fragment contains two RE site at each end. so if i want to ligate these two fragments just by B only, i have to consider A and C also whether they will self ligate. >

Trying to clone multiple fragments at one time is very tricky.  Try and put all three pieces into one ligation mix and go, but if it doesn't work after the first try or two, I would suggest coming up with an alternative strategy where you clone each fragment sequentially.  You will have a much higher chance of success.

Good luck,

J

If you do not want to mess with RE I would suggest getting Gibson assembly or golden gate cloning, works perfect ( I used it with up to 6 individual fragments) and it is not this expensive.

Good luck

add all the restricted inserts in double the concentration of vector and digested vector with A & C enzymes in a single reaction. Ligate at RT for 2-3 hours. You will get the desired product. Atleast 33% would be your product which will be good enough.

Ok Antonio, again, if you can invest some money (around 200 euros) go with In-Fusion or Gibson Assembly (have a look at the manuals I attach).

Otherwise, using what you most likely already have in the lab, go for fusion PCR (I attach a scheme for your understanding, and a link to another thread on fusion PCR within ResearchGate). Just keep in mind that when you set up the last PCR reaction where you fuse the two pieces, the first 5-10 cycles you run WITHOUT the primers (so that each fragment works as the primer for the other using the overlap region). This also means that you have to gel purify the first two fragments to avoid carrying on unwanted external primers. Then you add the external primers and run another 20 cycles.

In my experience, as others have pointed out, multiple insert cloning with REs is tricky and quite inefficient, that's why the seamless cloning strategies have been developed, as well as bacterial homologous recombination, which is anyway more complex and requires different bacterial cells expressing the recombinases that in standard cloning strains are for obvious reasons removed (check Fancis Stewart papers if you are curious about this technique).

https://www.researchgate.net/post/Can_anyone_help_with_fusion_PCR/1

You may combine the two inserts with overlapping PCR. Thus you simplify the problem to a two fragment ligation.

Hello,

I am sure, you have got enough ideas now. To sum up, the above answers, you have the option of:

1) use PCR to fuse the two fragments AB and BC together by having some overlap in between the two fragments,

2) keep trying your own method, with digested Vector AC and the two PCR fragments AB and BC, which I agree is going to be little difficult.

3) use some other protocol, as someone has mentioned Gibson assembly, which is same as the first one, that is also equivalent to SOEing reaction, trans-PCR, wherein you fuse two fragments owing to the overlap in them and this overlap can be something for 15-20 bp depending on the length of your individual PCR fragments.

4) This is what I would suggest, just do 1 PCR, either for AB fragment, or BC fragment. Just add the other RE to your primer. Lets say you plan to amplify AB fragment, then insert the restriction site C to the reverse primer, so your amplified product will look like ANNNNNNNN--------NBNNNNNCNNNN. The BNNNNNCNNNN are going to be the overhang in the PCR product. Clone this fragment in the digested vector AC as AC, then digest the resultant plasmid with BC, and clone your BC PCR Product. There should be some nucleotides between B and C to ensure proper digestion and make sure you do not end up creating any site that is present in any of the PCR fragment or vector.

You can do the same thing with BC fragment, but here your forward primer will have restriction enzyme A next to B.

5) You can have more alternate ways as well, have another intermediate cloning vector where either the restriction sites A and B or the restriction sites B and C are next to each other and then clone the products, one after the other and then later, take them out as insert AC and clone in the vector AC.

All the best,

Simran

Just add them all to the same tube and ligate.

Richard's suggestion should work, but might have relatively low efficiency. You might want to mix the two insert fragments and do PCR to combine them into one product. Use primers that anneal to the left end of fragment AB and to the right end of fragment BC. Use a low-error-rate polymerase such as Phusion by NEB. Then cut the PCR product with REases A and C and ligate to the vector.

Both the above ideas are good although the ligation might yield very low efficiency if added all three together. You can try doing an overlap extension PCR using primers having overlap regions of both the insert and then an extension like normal PCR with the end primers. This can be done using conventional PCR enzymes Taq or Pfu (NEB, Fermentas all works just fine). For details read the following paper: Nature Protocols 2, 924–932, 2007