I want to ligate a linear 1.7kbp plasmid with the EcoRI sites at two sides, but when I use the quick ligase kit from NEB to test the ligation efficiency it is hard to ligate it. Is there anyone who has the experience to do this experiment? and how can I optimize the experiment?
Ligationof a linear molecules with compatible ends towards forming a ring should be a very fast reaction, because it is an intrameolecular reaction, not requiring a second DNA partner. Normally, in intermolecular ligations, religation of the vector is by far the preferred reaction. Are you sure, you have no phosphatases around? Is your EcoRI pure? How do you check ligation efficiency? Do other reactions work with the same ligation mix?
Are you doing it at room temperature for 5 minutes? If so, try to increase the time. Good luck!
Hello Sam Gao ,
Try incorporating a positive control i.e. a digested fragment with the same sticky ends and check the extent of ligation with the same Ligase enzyme to ensure that the enzyme is effective. You can also try incubating the ligation mix at 16°C for 30 minutes or so even though it is a quick ligase.
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