Restriction endonucleases vary with respect to their ability to maintain activity in a reaction over an extended period of time.

1.0, 0.50, 0.25, and 0.13 units of restriction endonuclease were added to 50 µl reaction mixtures containing 1 µg of unit-assay substrate DNA. The reaction mixtures were incubated for 16 hours at 37°C (or as noted). The minimum number of units that resulted in complete digestion of 1 µg of substrate DNA in 16 hours was determined by agarose gel electrophoresis.

Enzymes that require less than 1 unit to digest 1 µg of DNA to completion in 16 hours can be used at lower concentrations for extended incubation periods. DNA substrates are digested at varying rates, the actual number of units required for a complete digestion in 16 hours will change from substrate to substrate.

Note: For enzymes that require 0.5 or 1 unit, overnight incubation will not improve digestion, and in some cases may be detrimental to the digested DNA. This is especially true for enzymes that cut away from the recognition site.