Restriction Enzymes 1293.4.2. Ionic Strength
The accuracy and activity of restriction enzymes are strongly affected
by the ionic strength of the reaction milieu. The required ionic strength
is achieved by the addition of NaC1 or KC1 to the Tris-HCl buffer.
Whereas most restriction enzymes accept both KCI or NaC1 some
enzymes strongly prefer potassium salts (e.g., SmaI). It must be empha-
sized that low ionic strength may induce star activity (e.g., BamHI, EcoRI,
EcoRV, XbaI), and that high salt concentrations may activate contami-
nant nonspecific endonuclease activities or inhibit the restriction enzyme.
Some suppliers (i.e.,AGS,Amersham, BRL, Boehringer, Stratagene)
have elaborated simplified buffer sets to be used with their enzymes.
Using only three different types of buffers, however, as recommended
for example by Davis et al. (18) (i.e., a low, a medium, and a high salt
buffer), does not meet the requirements of all enzymes and results,
therefore, in many cases in a lower specific activity. McClelland et al.
(181) and Pharmacia-LKB have developed one buffer system to be
used with all enzymes. The potassium glutamate buffer "KGB" and
the "One-Phor-All PLUS" buffer must be diluted before use according
to the specific requirements of several enzymes. Although the simpli-
fied buffer systems in general cannot be recommended for the eco-
nomic use of restriction enzymes, they allow the easy codigestion of
DNA with several enzymes. However, if different ionic conditions are
essential for site-specific cleavage with several restriction endonu-
cleases, the DNA should be digested consecutively, beginning with
the enzyme that has its optimum activity at the lower ionic strength.
When consecutive digestion is not possible because of incompatibility
of the reaction conditions, the fragments generated by the first cleav-
age reaction must be purified by phenol/chloroform extraction and
ethanol precipitation prior to incubation with the second enzyme. The
double digestion of multiple cloning sites in plasmid vectors with two
different restriction enzymes may cause severe problems; it is often
not possible to digest overlapping or directly neighboring restriction
sites in polylinkers.
3.4.3. Essential Cofactors
Mg 2+ ions are an absolute requirement for the catalytic activity of restric-
tion enzymes. Chelating agents, such as EDTA, therefore inhibit the
nucleolytic activity, whereas specific DNA binding is not disturbed (see