Reaction Conditions for Restriction Endonucleases:
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.
Updated September 18, 2002 15:00