Hi Praveen,

You could also digest the two PCR products with only XbaI and ligate them. Then take a small aliquot and do PCR again with the primers corresponding to the "new" ends. Purify the product and digest with the other enzymes then ligate it into the vector.

Good Luck!

Gisela

what i understood is that you have 2 fragments and you want to ligate them and then u want this large fragment to clone in the vector. If this is so then u ve to ligate the fragments first, do PCR to get on large fragment and then digest this large fragment at the ends, then do cloning in the digested vector

You might want to ligate the two fragments before adding it to your vector. That's how I've been doing it... and it has been working for my system. This way there is only one vector to insert.

Make sure that the ratio is correct... I would suggest using http://django.gibthon.org/tools/ligcalc/

as a guide for ratio.

Also, you might want to check to see if there is any Star activity in your initial digests (i.e. it is getting cut in different areas and leaving fragments).

how big are your inserts? did you treat vector backbone w/ AP? you could try ligating at lower temperature (maybe 4C overnight, or on ice)

A lot of colonies means you should decrease the vector concentration 10x at least. Increase the insert concentration if you have enough. Please do the negative control on self – ligated vector, since something is smelling not nicely out of this prep, what you probably noticed;)

Like Vanessa Selwyn has already said - do it in two steps: ligation of both inserts together and then ligation into the vector.

I would also add the cleaning step inbetween - if You isolate only the properly ligated fragment from the agarose gel (two inserts together), then You do not have non-ligated fragments binding to the vector and decreasing the efficiency of ligation. You will also eliminate some strange constructs due to the broken DNA or even the star activity of the restriction enzymes. You will lose some DNA during isolation from the gel, but the increase in the efficiency of ligation into vector will repay that.

Since undesired things can happen during ligation in standard conditions (vector+insert), even more can happen in your case (vector+insert+insert). I've never tried the two steps (ligate the two insert before, purify and then ligate into the vector) because after the first step you can't transform the (insert+insert) into bacteria to get more DNA. If you want to purify the (insert+insert) after first ligation you'll need quite a lot of material.

I think it could be better to try a two steps without purification (put the two insert together in the ligation mix, wait the manufacturer recommended time at the recommended temperature and then add the vector that you should have previously treated with AP, incubate again) and then after transformation, screen the colonies.

Anyway, to splice two DNA fragments I've always used overlap extension PCR:

http://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction

but it requires a different experiment design.

Do not forget to dephosphorylate your vector e.g. with antarctic phosphatase (NEB)!

Hi Reza,

I purified my digested vector and both pcr fragments and then put them all for ligation in ligation mixture containing T4DNA ligase. Then I transformed ligation mix and got many colonies but after screening of few of them, I found that only one insert or part of insert is ligated rather than both insert.

I agree with Imran. The more number of fragments are involved, the less likely they undergo correct positioning and ligation. I also suggest that after you mix all the fragments for ligation in a single tube, before you add the ligation enzyme and buffer, you heat the tube at 65 degree for 5 min and cool the tube on ice for 5 min. This allows for the removal of unwanted aggregation of the fragments which often happens during purification. Then, add the enzyme and buffer and ligate overnight at 16 degree. Then, use the whole ligate reaction to transform (check which strain of E coli is best for you. I routinely use DH5alpha or XL1Blue for transforming with large fragment.) You should have 5~10 fold higher number of colonies. Good luck.

Not to keep beating a dead horse, but... 1. Be sure your vector and PCR products are cut correctly and that you have cut and purified the proper bands. 2. Make sure your molar ratio of V:I:I is correct. 3. Dephosphorylate your vector. 4. Use an antibiotic resistant host strain like XL1-Blue/ tetracycline to reduce background colonies. 5. Make sure your media has the correct amount of antibiotics for your particular vector/insert and that they are not too old. If anything you should have too few colonies rather than too many when doing a sticky end ligation, particularly in a multi-part ligation. I have done up to 3 inserts simultaneously into a vector. Transformation efficiency is low when doing this, but if you spread your entire transformation on three or four plates, you should get your transformant.

Try Gibson cloning:

https://www.neb.com/products/e5510-gibson-assembly-cloning-kit

I now do this instead of 3 way ligation.

It is based on exonuclease chew back, annealing, resynthesis and ligation.

You just need PCR products with little overlap to vector and each other.

Works perfect to put 2 or more fragments together. Done this numerous times.

Hi All,

Thanks for all kind of suggestions.

I tried my old fashioned simple ligation and it worked for me. I just have to use good ratio of both inserts to vector ( both insert in molar ratio of 1:1) and transformed to E.coli ultra competent cells. Screening of 10 colonies by plasmid isolation and restricion digestion, confirms 4 positive clones.

Maybe you should check that your XbaI is able to digest fully DNA (for instance you can use a well-known plasmid to test its activity). Depending of the purchaser of the XbaI, I could not fully digest my fragments leading to cloning problems. Moreover, I am not sure where your restriction sites are located. For some restriction enzymes it is necessary to add certain nucleotide sequence on your primer in a way that your restriction site is not completly at the end of your primer and to favorise the digestion.

PEG8000 will increase the efficiency of ligation. You can try that some other time when your reaction fails.

Hi Praveen,

You could also digest the two PCR products with only XbaI and ligate them. Then take a small aliquot and do PCR again with the primers corresponding to the "new" ends. Purify the product and digest with the other enzymes then ligate it into the vector.

Good Luck!

Gisela

Hi Praveen,

If you are trying to clone two pcr products on a vector, I think that you should anneal the pcr products in a SOE pcr. In order to do this, you have to add complementary ends to the internal pcr primers. Once you obtain the annealed product, you can digest and clone or clone in a cloning plasmid.

Revisa las concentraciones de tus reacciones de restricción y ligación; el buffer adecuado es importante ya que si alguna enzima que usas tiene actividad estrella eso ocasiona problemas. Usa la cantidad de enzima recomendada. También verifica que tus enzimas y reactivos estén libres de contaminación. Si purificas por extracción en gel de agarosa y usas transiluminador con luz UV, (verifica que la longitud de onda que usa el instrumento sea la correcta), se rápido pues se daña el ácido nucleico y esto también ocasiona problemas.

Some good answers above. You might want to try increasing the concentration of inserts in order to "force" the three-way ligation. Alternatively, you could also try a modification of PCR-ligation-PCR cloning that we invented back in 1995 (see Biotechniques 18(5) 746-50). Here, you would ligate your XbaI digested fragments, then PCR amplify using the upstream primer of the first fragment, and downstream primer of the second. BamHI/KpnI digestion would then generate the single insert you require.

Good luck! Stuart

Whatever you do, you run the risk of self ligation of the inserts since you are not dephosphorylating the inserts. This effectively reduces the number of the right type of ligation products available to ligate to the vector. Using a 1:1 molar ration is your only hope. From your answer above, it looks like that strategy has worked.