Most restriction enzymes work acceptably in a pH range between 7.2 and 8.0. The activity and specificity of restriction enzymes are strongly influenced by the salt conditions or ionic strength of the reaction mixture. The ionic conditions in the multiple digestion reaction mixture must be chosen so that the activities of both enzymes are high. Usually, this requirement can be fulfilled by adding an appropriate concentration of either KCl or NaCl to the reaction buffer. A few enzymes, however, require potassium and will not work in the presence of sodium.
Ionic strength can also influence the specificity of the restriction enzyme.
For instance, low ionic strength has been shown to induce star activity with enzymes such as Bam H I , Eco R I and Sph I . Reaction temperature is a very important parameter that will determine the success of the restriction digestion. The catalytic activity of enzymes is significantly influenced by temperature. Most enzymes require 37 C incubation temperatures for optimal activity. Other enzymes require higher or lower temperatures.