Assays for Restriction Endonucleases Using Plasmid Substrates

A type II restriction enzyme purchased from a commercial supplier comes with a specified number of units of enzyme activity. The units of restriction enzyme activity are defined by the minimal amount of enzyme needed to complete the digestion of 1 l.tg of bacteriophage γ DNA in 1 h. These units are usually measured by making serial dilutions of the stock solution of the enzyme, adding 1 PL from each dilution to 1 �g of phage γ DNA in a suitable buffer, incubating the reactions for 1 h at 37�C, and then analyzing the DNA by electrophoresis through agarose. This is, at best, a semiquantitative assay. It cannot yield quantitative data about the rate of the reaction of a restriction enzyme on a DNA substrate.