Methods in Molecular Biology • 16 Enzymes of Molecular Biology

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BAL 31 Nucleases 241


by overnight stirring. NaOH (2M or higher) is added to a nominal
concentration of 0.1M and, after several minutes of alkaline denaturation,
the solution is neutralized with a severalfold excess of 3M sodium
phosphate (pH 7). Dialysis into BE buffer is followed by adjustment
to the proper concentrations of NaC1, MgC12, and CaC12 in 20 mM
Tris-HC1/1 mM EDTA (pH 8) to yield, after addition of enzyme solu-
tion, the desired composition of assay buffer: 0.6M NaC1, 12.5 mM
CaC12, 12.5 mMMgC12 in the aforementioned Tris/EDTA buffer. If0.1
vol of enzyme solution in CAM buffer (100 mM NaC1, 5 mM MgC12,
5 mM CaC12, 20 mM Tris-HC1, 1 mM EDTA [pH 8]) is mixed with the
DNA solution in the assay, the DNA solution should contain 0.656M
NaC1, 13.3 mM MgCI2, and 13.3 mM CaC12, which is conveniently
achieved with 5,0.5, and 0.5M stock solutions of the respective chemi-
cals, each containing the Tris/EDTA buffer. CAM buffer (usually
containing 50% [v/v] glycerol) is used as the nuclease storage buffer
by several suppliers.
After mixing 0.9 vol of the above DNA solution with 0.1 vol of enzyme
solution at 30°C, aliquots of 0.4 mL are withdrawn at such times as 5, 10,
and 15 min and mixed with 40 IlL of 0.5M sodium EDTA (pH 8-8.5)
followed by 0.8 mL of 10% (w/v) HCIO4. This is conveniently done in a
1.5-mL Eppendorf centrifuge tube for centrifugation of the precipitated
nondigested DNA after the acidified mixtures have stood on ice for sev-
eral minutes (but not for extended periods). Five-tenths to 1 mL of super-
natant is carefully removed from each tube after centrifugation for approx
10 min, and the value ofA260 is measured in a 1-cm path length quartz
cuvet with a 4 mm wide cavity. The blank (zero time of incubation) is
made up separately by mixing 40 IlL of 0.5M EDTA with 0.4 mL of
the denatured DNA solution before addition of the enzyme and HC104
solution. This ensures that any acid-soluble UV-absorbing material in
nuclease preparations will not contribute to the A260 values when the
blank absorbance is subtracted.
The resulting plot of A260 vs time (t) (in minutes), including the
point (0,0), should be a good straight line (correlation coefficient from
linear least squares analysis 0.995 or greater) from which the value of
the slope, AA260/t, is obtained. According to Vogt (32), the number of
enzyme units in the total mixture is given by:


U = (AA26o]t)(30.3)(Vtot) (1)
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