Dear researchers,
I have performed a cloning study for a gene 3550 bp but it didn't work. Firstly I gel-extracted the gene after digesting the vector with two restriction enzymes. I have also digested the recipient vector with the same enzymes and gel extracted respected band. I have checked insert and vector purity and band size in agarose gel and they were looking at expected size single bands. I have tried 1:1, 2:1, 3:1 ratios for ligation with NEB T4 DNA Ligase with the manufacturer's instruction. I have transformed the ligation product into E. coli XL10 but there are no colonies. I guess I am stuck in the ligation ratio. Which ratio I must use? Or is there a different ligase option that you newly know about?
Small differences in insert to vector ratios are not going to have much of an impact. So the fact that you did not get any colonies suggests that there is some other problem and not the ratio.
It could be a problem with your vector, maybe it was UV irradiated too much when doing the gel extraction. Or you lost too much during those steps. Or it could be that your competent cells are not good enough for a cloning.
I would run appropriate controls and check the different steps, but I am confident it is not the ratio.
Thank you for your reply Dr. Benedik. I have considered all possible risks. And I had freshly prepared competent E. coli XL10-Gold cells. The vector seems okay because I have a few colonies due to ligation and transformation at a ratio of 1:1. As a result of colony PCR, I noticed that the vector closed on its own without receiving the insert. Today, I remade the insert and ligated 1:1, 3:1, 5:1, and 6:1. I'll see the result tomorrow. I will consider your suggestions.
I agree with the irradiation. You can also try PCR vector and insert with some overlapping between them separately and then try Gibson assembly. Use vector only as a control and see if it helps.
Thank you very much Dr. Quedez. We have just got a new machine in that I performed gel extraction without UV light. I will try again if it works I will share my optimized protocol here.
Finally, we succeeded in cloning. We confirmed that the problem was not in the ligation ratios, with the positive PCR results of the colonies formed at the ratios of 1:1, 3:1, and 7:1. The problem was in our enzyme. We have overcome this problem by using the new NEB T4 ligase enzyme.
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