What are conditions for ligation?

Firstly: How did you know that your ligation isn't working? Did you check whether each of the restriction sites of your plasmid is cutting appropriately? If yes, then how long and under what temperature did you perform your ligation? Buffers for T4 DNA ligases are usually supplemented with ATP. If they are not supplemented in your case, then be sure to add them separately. Also, care should be taken when you are adding the buffer as ATP is susceptible to degradation upon frequent freeze-thawing. Try using 1:5 ratio as well. You can also go up to 1:20. Good luck

Are you purifying the digested fragments? I suggest that you run the digests on agarose gel and purify the fragment with Gel Extraction kit by Qiagen. Then, determine the concentration of each purified fragment and mix vector and insert at the optimal molar ratio (1 vector to 3 fragment) to the total concentration of ~100 ng/10ul reaction. Before you add the ligase and buffer, you can heat the tube at 65 degree for 5 min and cool it on ice so that you can remove unwanted aggregation of DNA. After you add the enzyme and buffer (I usually use 1 ul each in 10ul reaction), you can let it react at 16 degree overnight. Then, use the whole reaction to transform one tube of bacteria (the volume of competent cells should be >100 ul). Try this and it should work. Good luck.

BamHI and HindIII double digests work very well, I have used them several times. You dont need an alkaline phosphatase treatment in double digests. But you must gel-purify the digested insert/vector as Saori says. I am sure you checked the digested insert/vector on a gel quickly to know that the digestion is good. Usually its the ligation which is a problem, and if you use gel-purified vector/insert with a fresh ATP stock, it should work. Finally, you may consider changing to a fresh T4 ligase vial.

Hi Sudeep,

if your BamHI and HindIII sites in you plasmid are very close to each other you should first digest with BamHI and afterwards with HindIII. This is neccessary because BamHI will need at least a 5bp overhang 3'- and 5'- of the GGATCC site to bind and cleave properly. I am not sure about HindIII, but BamHI will need this overhangs definately. The same is important, if you added the BamHI sites to you insert by PCR as well. (http://www.ncbi.nlm.nih.gov/pubmed/11254386)

If you are using BamHI and HindIII for directional cloning you should be able to skip alkaline phophatase treatment since the sticky ends produced will only fit in the correct way and religation of the plasmid should not accure if the cleavage worked well.

Best Kai

I have checked the restriction digestion through agarose gel and restriction sites are 5000 bp apart and I do gel purification with Qiagen kit. I have tried without alkaline phosphatases also and as well as 1:5 ratio. I used fresh Ligase and buffers too. But I couldn't get colonies.

I will try for 1:20 ratio and also heating them up to 65'C.

16'C overnight is usual protocol for ligation which I follow.

Hi there,

I had such a cases in my lab when I was transforming Ecoli. Make sure, that your ligation mixture does not contain any restriction enzyme left. Because even small amounts of active RE can destroy your experiments. It is obvious, but still, sometimes we do not remember about it. You have to eliminate all the RE that could still be attached to the digested DNA. So, you can do this simply by: performing thermal inactivation of used RE (or phenol/chlorophorm extraction), or perform gel electrophoresis of the restriction mixture and extract THE band of interest. Then do the same with the vector (add AP), extract DNA, ligate the two DNA molecules (overnight, 4C) and do the transformation. It will be the best to use the same buffer for all the enzymes used in the experiment.

Good luck!

Hi!! Did you check the concentration of your Insert and Vector? which vector your are using for the cloning? check the concentration on nanodrop and running in the gel as well..how much reaction volume you are using for the ligation reaction??

Sudeep, I wanted to find out the following: when you talk about insert:vector ratio, do you mean molar ratio?

Are you getting colonies with your vector-only control ligation? What about the vector-only control if you do not treat the restricted vector with phosphatase?

When trouble shooting a problem like this, just check each component. (1) make sure your competent cells are working well (2) make sure you actually have DNA after gel purification (3) make sure your ligase and buffer are still working. Given what you wrote, I would first check your competent cells.

I had such a case in my lab, too, which blamed to the fake agarose. The impure agarose contains multiple impurity polysaccharide, which blocks the down-steam manipulation intensively, including amplification, ligation, digestion etc. . The subsequent experiment employed agarose purchased from invitrogen were carried out successfully.

I have checked my competent cells with other complete plasmid, they got transformed and formed colonies. I have checked the concentration of plasmid and insert after digestion and gel purification; used 1:1-1:10 ratio of vector vs insert ratio. For more confirmation I borrowed enzyme and buffer from other working lab they also didn't work. So I have ordered new enzyme and buffer to perform all the suggested protocols . I will be thank full of you all for your kind suggestions and I still would like have more suggestions and quarries about my mistakes.

Maybe you should try another restriction site, or try a new plasmid.

Good luck!