Most of the restriction enzymes have an optimum temperature of 37°C and an optimum pH of pH 7.2 or pH 8.5. These physical environment requirements must be met to ensure that the enzyme reaches its maximum activity. Incubation temperatures affect the activity of restriction enzymes greatly. The enzyme can denature outside the desirable physical condition range. Enzymes work very specifically; therefore, they will not work properly if their structure deteriorates. As each restriction enzyme is isolated from different bacteria and have different optimum reaction conditions, they require specific buffers for each restriction reaction.

Table 1: Enzyme activities. The number for each buffer signifies the activity in the respective enzyme. If there is a * the enzymes may exhibit star activity in this buffer.

Enzyme Sequence Buffer1 Buffer2 Buffer3 Incu. Temp.
EcoRI G'AATTC 25 100* 50 37°C
KpnI GGTAC'C 100 75 10 37°C
PstI CTGCA'G 75 75 100 37°C
SpeI A'CTAGT 75 100 25 37°C
XbaI T'CTAGA 10 100 75 37°C
XhoI C'TCGAG 75 100 100 37°C