Restriction Enzymes 163
note added in proof at end of chapter). Aconcentration of 5-10 mM MgC12
seems to be optimal. Some enzymes (e.g., NlaIII and NlaIV) additionally
require 50 mM (NH4)2SO 4 to be activated.
3.4.4. Stabilizing Additives
Stabilizing additives, already mentioned as components of the stor-
age buffer (Section 3.1.), are used to maintain the activity and accu-
racy of restriction enzymes during the reaction.
3.4.5. Reaction Time and Temperature
The reaction time needed to digest a given amount of DNA is deter-
mined by the activity of the restriction enzyme. However, it is not
useful to exceed the expected lifetime of a restriction enzyme under
the reaction conditions indicated in Table 5. The repeated addition of
the required activity may overcome problems concerning the extreme
instability of some enzymes (e.g., ScaI, SphI, StyI, CfoI). Care has to
be taken that accumulation of glycerol caused by the repeated addition
of enzymes does not induce "star" activity.
Temperature is a critical parameter for the optimum use of restric-
tion enzymes. Whereas most restriction enzymes have temperature
optima around 37°C, some enzymes, in particular those isolated from
cryo- or thermophilic bacteria, need relatively low or high tempera-
tures, respectively, for catalytic activity. The optimum incubation tem-
peratures are summarized in Table 5. For incubation at elevated
temperatures, one should seal the reaction vessels or overlay the reac-
tion mixture with parafine oil.
3.5. Assays for Purity
of Restriction Endonuclease Preparations
Commercially available restriction enzyme preparations are nor-
mally tested by the suppliers for the absence of the following contami-
nants: other specific restriction endonucleases, nonspecific
endonucleases, 5'-exonucleases, 3'-exonucleases, and phosphatases.
The absence of these enzyme activities can be assayed by the frag-
ment-pattern-stability assay, plasmid-nicking assay, 5'-exonuclease-
phosphatase assay, 3'-exonuclease assay, and ligation-recut assay (see
the following sections). Depending on the specifc application, the
absence of contaminant activities is more or less critical: Whereas