Thermal Inactivation

Thermal Inactivation

A simple, reversible way to a stop restriction reaction is by adding EDTA, which chelates Mg²⁺ , thereby preventing catalysis. If further manipulations of the digested DNA are to be performed, the restriction endonuclease should be inactivated. Phenol/chloroform extraction and ethanol precipitation is an irreversible method for inactivation and removal of all restriction endonucleases; however, a more convenient method is thermal inactivation. Most restriction enzymes can be inactivated by incubation at 65°C for 20min. Others remain active at 65°C but lose their cleavage ability at a higher temperature. Even many thermophilic enzymes that show optimal activity at 50-55°C can be inactivated at 80°C in 20min. Information on the susceptibility of Fermentas restriction endonucleases to thermal inactivation and the temperature required to achieve this is presented in the table  "Reaction Conditions for Restriction Endonucleases ".

Conditions.   10-100 units of enzyme were incubated at optimal reaction conditions for 1 hour with 1µg of appropriate DNA substrate (usually plasmid DNA containing at least one recognition site for the restriction endonuclease tested). After incubation at 65°C or 80°C for 20min, 1µg of control DNA (lambda or Ad2 DNA used in standard unit determination reaction) was added to the reaction mixture and incubation was performed at optimal temperature for a further 60min. Reaction products were analyzed by agarose gel electrophoresis. The absence of subsequent substrate cleavage was interpreted as thermal inactivation of the restriction enzyme.