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

While most routine restriction digests are incubated for one hour or less, there are certain applications that require the addition of less than 1 unit/µg of DNA and increasing the reaction time beyond one hour. The table below can be used as a guide when low levels of enzyme and extended reaction times are needed.

For example, 1 unit of Aatll can be used to digest 8 µg of DNA in a 16 hour digest (+ + +).

Extended activity was determined by performing the restriction endonuclease unit assay, using a 16 hour incubation in place of the standard 1 hour digestion. After the 16 hour digestion, extended activity enzymes (+ + +) required only 0.13 units to completely digest 1 µg of DNA. Intermediate activity enzymes required either 0.25 (+ +) or 0.50 (+) units for complete digestion over this extended incubation time. Finally, enzymes marked (–) required 1.0 unit for complete digestion, the same amount of enzyme required for a 1 hour digestion.