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Restriction Enzymes Resource 

2.2 Restriction Enzyme Activity

Restriction enzymes differ in their reaction kinetics. As a result, variations from the recommended incubation time, number of units used, substrate amount, and/or total reaction volume should be considered carefully to ensure complete digestion. The following table gives an indication of the activity of Promega’s blue/white cloning qualified and genome qualified restriction enzymes under varying reaction conditions. Variations in the number of enzyme units used and the reaction incubation times were tested. In each case the reaction volume (50µl) and the amount/type of DNA substrate (1µg) were the same as that used in the unit definition assay. Incubation time for the unit definition assay is one hour.

Table 2.2. Restriction Enzyme Activity under Nonstandard Units and Incubation Time Conditions.

Enzyme

Reaction Time and Number of Units Used

15 min.
4 units

15 min.
2 units

15 min.
1 unit

30 min.
2 units

1 hr
1 unit

2 hr
0.5 units

4 hr
0.25 units

Aat II

C

I

I

C

C

C

C

Acc I

C

C

C

C

C

C

I

Acc65 I

C

I

I

C

C

I

I

Apa I

C

I

I

C

C

C

C

Ava I

C

C

I

C

C

C

C

BamH I

C

C

I

C

C

I

I

Bbu I

C

I

I

I

C

C

C

Bcl I

C

I

I

C

C

C

C

Bgl I

C

I

I

C

C

C

C

BstX I

C

I

I

C

C

C

C

BstZ I

I

I

I

C

C

C

C

Cla I

C

I

I

C

C

C

C

Csp I

C

I

I

C

C

I

I

Csp45 I

C

I

I

C

C

C

C

Eco52 I

C

I

I

C

C

I

I

EcoR I

C

I

I

C

C

C

C

EcoR V

C

I

I

C

C

C

C

Hinc II

C

I

I

C

C

C

C

Hind III

C

I

I

I

C

C

C

Kpn I

C

I

I

C

C

C

C

Mlu I

C

I

I

C

C

C

C

Nco I

C

I

I

C

C

C

C

Nhe I

C

I

I

C

C

C

C

Not I

C

I

I

C

C

C

I

Nsi I

C

I

I

C

C

C

C

Pst I

C

I

I

C

C

C

C

Sac I

C

I

I

C

C

C

C

Sac II

C

I

I

C

C

C

C

Sal I

C

C

C

C

C

C

I

Sfi I

C

I

I

C

C

C

C

Sma I

C

C

I

C

C

C

C

Spe I

C

I

I

C

C

I

I

Sph I

C

C

C

C

C

I

I

Ssp I

C

I

I

C

C

C

C

Sty I

C

I

I

C

C

I

I

Xba I

C

I

I

C

C

C

C

Xho I

C

I

I

C

C

C

C

Xma I

C

I

I

C

C

C

C

"C" indicates complete cleavage; "I" indicates incomplete cleavage for the units and incubation times shown.

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