I am using fermentas T4 DNA ligase enzyme for ligate binary vector with 2.7kb insert. But it does not show any positive colonies. How can I check that enzymes is working?.
For checking whether the T4 ligase is working or not ...you can do a very simple experiment......use DNA markers available in the lab...(PhiX 174 digested with HaeIII or lambda DNA digested with HindIII or any other marker)....and ligate 100 ng of it in a standard ligation reaction.....after the recommended time...run the ligated marker along with the standard marker side by side on an agarose gel....you'll see how effectively ligase is working......Generally if you are working a rapid DNA ligase/kit...you can see >90 % ligation within five minutes or so...