164 Pingoud, Alves, and Geigervery pure enzyme preparations are needed for DNA cloning, digestion
of genomic DNA for Southern analysis is not impaired by a contami-
nant phosphatase activity.
3.5.1. Fragment-Pattern-Stability Assay
Contamination of enzyme preparations with other specific endonu-
cleases is assayed in ~. DNA cleavage experiments as described above,
however, with incubation periods of up to several hours. The absence
of a contaminating site-specific endonuclease can be verified by exa-
mination of the generated ~.-restriction-fragment pattern. It should be
kept in mind, however, that the prolonged incubation of a DNA sub-
strate with a large amount of a restriction enzyme preparation may
produce an altered banding pattern because of the limited accuracy of
all restriction enzymes. The sites cleaved under these conditions are
normally the same as those that are attacked under "star" conditions
after shorter digestion times. Nonspecific nuclease contaminations
cause degradation of the individual fragments to yield a "smear" on
the agarose gel.
3.5.2. Plasmid-Nicking Assay
The absence of contaminating nonspecific endonucleases may be
assayed by incubating any supercoiled plasmid or phage DNA that
does not harbor a site for the specific endonuclease for several hours.
The cleavage of a single phosphodiester bond in the superhelical DNA
results in a conversion to open circular DNA, which can be easily
detected by electrophoresis on agarose gels containing 0.01 mg/mL
ethidium bromide.
3.5.3. 5'-Exonuclease / Phosphatase Assay
To detect any contamination with 5'-exonuclease or phosphatase
activities, the following standard assay can be used. ~, DNA is com-
pletely cleaved with a restriction enzyme producing a large number of
fragments with 5' protruding ends (e.g., HpalI, HaplI, MspI). The
DNA fragments are labeled usingT4 polynucleotide kinase (see Chapter
20) and "l([32p]-ATP and ADP in an exchange reaction. The resulting
labeled DNA fragments are purified by chromatography over DE52
spun columns. The radioactively labeled DNA is then incubated with
the enzyme preparation to be tested. During this incubation, any contami-
nation that liberates phosphate or mononucleotides from the 5' end can