Hi Fazlur,

Usually I do not run the ligation on a gel, since you would expect the plasmid to be circular, hence it would not necessarily reflect the real size of your construct or your insert. Can you give me some more details on what kind of ligation you are trying to do? There could be a number of reasons why your cloning does not work. Do you have sticky ends or blunt ends? If you have blunt ends, did you dephosphorylate your vector? Do you get the same number of colonies on your vector only (negative) control? Which ligase are you using? Are your media plates fresh? Cheers Simone

Hi,

Going only with what you have described and not knowing the detals of your ligation, I suspect that one of the ends of your insert is faulty, which may be why you get a band twice the size of the insert, without a trimer or tetramer. Like Simone above, I need more details before saying anytihing more helpful.

Hi Simone and Ken, thanks for reply, I have sticky end with Bg1II and Sal1..I dephospyorylate my vector...that's why I am not getting background (only 2-4) colones in entire dish..I get the same no fo colonies in vector only and the ligation product..I feel  like it is not ligating..I cut an insert from my vector..so no reason that the both ends are not cut..again for insert I used the same enzymes..so dont understand why it would not work..but like Ken said, im getting just dimer but not multimer..there should be a dimer of vector too right? but I am not getting vector dimer either..please help..oh I am using T4-ligase..i would assume ligase is working since I see dimer of insert right?

...i did a control ligation of my insert and checked.. i see the trimer which is almost same size as the vector..so no problem with the insert cut at both side..but i dont see any band for vector dimer..am I supposed get vector dimer? or ligation ratio is the probem?

So it is not a problem with the restriction enzymes or the ligase it seems. Have you tried using different molar ratios of vector and insert? Also incubating the ligation reaction at different temperatures might help (16˚C over night works well for me). 

since I dephosphorylate the vector..i should not get dimer of vector right? I think it is something with ratio and temperature..i will try different ratio and temperature and let you all know how it works..thanks

Dear Fazlu,

Just a thought occurrd to me. Is your BglII and SalI sites next to each other in your vector? If they are, what can happen is that when the vector is cut by one enzyme, it can no longer be cut by another because of insufficient space for the second enzyme to sit on and carry out the second site. In this scenario, the vector will have only one cut and will not be able to be ligated with your insert with two different restriction sites.

Let me know how many bases apart are the BglII and Sal sites are on the vector.

Ken

Hi Ken, they are 40bp apart

Hi Fazlur,

You blew that possibility out of the water. 40 bases is plenty for restrcition enzymes to sit and cut. I have more question though. After dephosphorylation do you clean up your vector? If you do how do you do it? Do you use a column or run through a gel and gel extract? Although it is usually fine, sometimes, agarose can carry over and interfere with ligation. Furthermore, which alkaline phosphatase do you use and do you inactivate it? This enzyme tends to be hard to kill and can stick around if it is not fully inactivated and removed and this will inhibit your liagtion.

In addition to this, have you tried to ligate your vector after cutting without first dephosphorylating the ends? If you try this and it works you will get multiple bands bigger than the linear length of the vector. Then you know that the probelm is down stream and you could then start by considering the issue with alkaline phosphatase as described above and move your way down the protocol. So try the ligation without dephosphorylation first.

Ken

Hi Ken thanks a lot..after dephosphorylation I clean up by running it on gel..i clean up running through gel in every step..i checked the insert (which i purified by gel after digestion) only ligation and saw dimer trimer..i did not check the vector before phosphorylation..but I always get ligation successful with gel purified vector except this case..i am using promega alkaline phosphate and i never needed to deactivate it..now im trying to cut the vector again (this vector has a big insert) just to make sure that my vector is cut properly..the only reason i did not use it earlier bcoz this insert is same size with the new one im introducing and make it hard for me to evaluate the positive clone..but will see what happens..i will do vector dimerization assay..thanks..i will keep you posted

Hi, Fazlur, You may want to try single digestion by each of the enzyme and check if both of them could linearize your vector. 2. you should also put a positive control in your system when ligation did not work, eg. single digestion and ligation. 3. to my experience, double digestion did not require de-phosophorylation if the digestion is complete. Adding an addition de-phosophorylation step may increase the difficulty of trouble shooting. 

hi Ken, I get the vector dimer trimer..then i did dephosphorylation and purified with spin column..hope the ligation works..i should a band above the vector right? i never seen yet...hopefully i see band..trying different ratio

hi Ken, i see my insert forms multimer and vector forms multimer..so no issues with digestion..but still no vector insert ligation product..it is driving me crazy..what do you suggest? on the gel of ligation product, I see the multimers of insert and vector has the same band intensity with the control..what can I do..i tried different ratio and different ligation temperature..should I try with our dephosphorylation of vector? oh..i did spin column to purify vector afeter dephosphorylation..

Dear Fazlur,

Sorry for the delay in replying. I am not very clear about what you described. You said you see multimers of insert and vector. Multimers of insert is good and expected, but multimers of vectors is unexpected if the vector was dephosphorylated. So, when you say this, do you really mean that the de-phosphorylated vector alone become multimer after ligation?

Between the vector and insert, I think the probelm is with your vector. Have you tried and if not, can you try the following:

1) Prepare three digestions: One with BglII only, the second with SalI only and the other with BglII and SalI. Run the digestions and see if the all correxpond to the expected sizes

2) If the above is OK, carry out two co-digestion of BglII and SalI in two tubes. One tube, add phosphatase, the other, do NOT add phosphates. Purify both separately through your usual spin column. Setup ligations and run the samples on the gel to see what you have.

When you have done that let us know and we can see what you get and from there make suggestions of the next step.

Good luck

Ken 

hi, I see multimers of vector if I set up vector only ligation before phosphorylation..and after phosphorylation no multimer of vectors..so no problem with digestion..digestion is 100% right for vector and insert..i think it is one of those following two happening..1)the dephosphorylation causing something to vector 2) the insert is doing something to the vector....what can dephosphorylation do to the vector so that it cannot ligate any more? any suggestion..? if I proceed with ligation without vector dephosphorylation I will see vector multimers and insert multimers and will be hard to explain the ligation right..

Hi, Fazlur,

Thanks for your reply. I see the situation now. Yes the problem is probably the phosphatase. I presume you gel-purify your insert right? If you do, then is it possible to gel-purify your treated vector as well. What I am thinking is that the phosphatase might still be bound to your DNA DNA and does not get removed during column purification. However, by running it through the gel, this might be achievable. This is just speculation. I have no references to support this, but I have found gel-purified vectors to give me better results. Admittedly, I have not done very careful comparison. Just a semi-qiantitative observation.

Alternatively, you can forget the dephosphorylation altogether and carry out the cloning wthout de-phosphorylating the vector. You will not have much problem of religation because the BglII and SalI sites are non-compatible so will not anneal. Although multimers of vectors can form, they are unstable because they have multiple origins of replication and they are not efficiently maintained in the bacteria. So most of the colonies that you have will be those with the insert cloned. I would try the this if I were you.

Good luck

i gel purified my vector once before..it did not help..i think I will proceed without phosphatase..lets see what happens..is there any way to confirm the ligation success by running on the gel if i did not dephophorylate vector?

Hi Fazlur,

You seem to have tried almost everyhting. I feel your frustration. So let us hope that ligation with non-dephosphorylated vector will give you success. I would not check the ligation reaction. I would just transform bacteria and plate them and see if the number of colonies in the vector+insert is greater than the vector alone. This is fast and efficient. The other reason for doing this instead of checking on the gel first is that ligation products form many bands that are very difficult to interpret because circular DNA with nicks run slower than their actual size while their supercoiled forms run faster than their actual linear size. These species will be accompanied by multimers of the inserts and multimers of vectors and multimers of both, and all with the possibilities of linear and circular. So when you get a series of bands of varying sizes, it is firstly dicfficult to know which and corresponds to which species of DNA but secondly, it is good news that one of the bands is the circular vector + insert. That is why the simplest way to know is to transform the bacteria and count the colonies and pick a dozen and do mini-prep to look for the positive clones.

Let me know how you get on.Good luck 

thanks a lot..i will do that..hopefully it will work..also i don't need to inactivate ligase before transformation..do you suggest inactivating it?

Hi Fazlur,

There is absoltely no need to inactivate ligase. I have never done this in 20years of cloning.

Good luck

Thanks..I let you know how it goes..

Hi Ken, I did the ligation without the phosphorylation..when I checked it on the gel..I got smear in the ligation reaction which I could not explain..then I transformed and plated..i got less colonies in the ligation product compared to vector only control..should I still proceed and do the miniprep and check.? let me know your advise..thanks.

Dear Fazlur,

Sorry for the delay in replying. I am away trekking in India at the moment. Very strange, but it is worth doing miniprep of some of the colonies to see what you get.

Let me know what you get and oh, I do not know when I will get internet connection, so no guarantee that I can reply promptly.

Good luck

Ken

Hi Ken, I screened the colonies..no luck..I don't know what else to do..

It is not necessary to run the ligation reaction mixture on gel as the amount of the ligated insert and vector may be too low to be detected by gel electrophoresis, which can still give you successful transformation.

Additionally, there will be ligase attached to the DNA which may result in uneven running pattern on gel which may not reflect atcual size of the DNA.