128 Pingoud, Alves, and Geigergels, whereas complete EcoRV-~,-fragments (ranging from 270-5770
bp) require separation on at least 1.5% agarose gels (see Table 4).
Volume activity (see Section 3.2.) toward ~, DNA is calculated by
determining the lowest enzyme dilution that allows complete cleavage
of all recognition sites to yield the characteristic cleavage pattern for
the particular enzyme.
It should be kept in mind that the volume activity measured with ~,
DNA does not directly indicate how much enzyme is required for the
complete cleavage of another DNA substrate. Apart from influences
concerning DNA conformation and flanking sequences discussed
above, it is necessary to consider the concentration of sites for each
substrate. For example: ~, DNA with a total length of 48,500 bp has
five EcoRI sites, i.e., 1 site/9700 bp, pBR322 with a total length of
4363 bp harbors only one EcoRI site, i.e., 1 site/4363 bp. Therefore, in
pBR322, there are 2.2 times more EcoRI sites/lag DNA than in ~, DNA
and more enzyme will be required. The exact amount of additional
enzyme will depend on the kinetic properties of the enzyme.3.4. Optimum Reaction Conditions
The specific activity and accuracy of restriction enzymes mainly
depend on appropriate reaction conditions with respect to pH, ionic
strength, cofactors, stabilizing additives, reaction time, and temperature.
Optimum reaction conditions are normally indicated by the enzyme sup-
pliers. Table 5 summarizes reaction conditions regarded as optimum for
the commercially available enzymes.3.4.1. Buffer pH
Whereas most restriction enzymes work well in the pH range from
7.2-8.0, some enzymes have more narrow pH optima. For example,
the activity of AatlI significantly decreases outside the pH range 7.4-
7.8. In several cases, it has been observed that high pH (>8.5) may
induce star activity (e.g., EcoRI, DdeI). The recommended reaction
pH refers to the optimum reaction temperature. The pH of the reaction
buffers, therefore, should be adjusted at this temparature. Often the use
of a 20-50 mMTris-HC1 solution is recommended as buffer. The Tris-HC1
system, however, is markedly temperature-dependent because of a ApKa/
°C of-0.031, which results in a pH shift of approx 0.25 pH U/10°C.
Also, not all pH electrodes are suitable for use with Tris buffers.