Hello All. I am trying to ligate the blunt end of maize genomic DNA digested by restriction enzyme. I do not have the option of using restriction enzyme with sticky end cuts.. I am using T4 DNA ligase from Thermofisher scientific and following their protocol but never worked. Any idea will be highly appreciated.
Thank You
A few thoughts come to mind to consider: 1) do you know that the ends of the DNA are truly blunt? Sometimes shearing leaves ends that are missing one or two bases and are not really blunt. You might need to polish the ends to get efficient ligation. 2) do you know your enzyme and especially ligase buffer are fresh and working well. If your stock is old, the ligase buffer may be off. 3) Is your DNA concentration ok, optimal conditions for circularization are low DNA concentrations. At higher concentrations you will drive the reaction towards ligating different fragments together.
Hi Michael, the ends are blunt, I used Hin1ll restriction enzyme. Ligase buffer is knew and working for sticky ends. I will try reducing the concentration.thank you.
Hin1ll does not generate blunt ends, it generates a 4 base CATG overhang. However it should ligate even better.
Have you monitored on gel whether you are getting some mobility shift of your DNA fragments after ligation?
Sounds like you may have a ligation issue then, either with the enzyme or the buffer. I presume you are inactivating or removing the restriction enzyme before the ligation.
Yes I inactivate the restriction enzyme before ligation and then proceed to ligation. However I do not deactivate ligase..
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