What molar ratios should I use for DNA Ligation?

The optimal reactant ratio is contingent upon the downstream application.


Eric Cantor:

General subcloning of a sticky or blunt insert into a vector is best accomplished with an insert to vector molar ratio of three to one. This ensures that only one insert is ligated into the vector. Lower insert amounts reduce the ligation efficiency and higher amounts increase the likelihood of multiple insertion events. Adaptor ligation onto DNA fragments benefits from higher ratios ranging from 10 to 1, to 50 to 1 because circularization is not desired. Circularization of single DNA benefits from lower concentrations to reduce the likelihood of intermolecular ligations. These considerations are more critical than the actual choice of reaction volume.

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