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Survival in a Reaction

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 (defined for each enzyme in the Restriction Endonuclease section of our catalogue). 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.

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