Deoxyribonucleases of Molecular Biology 13
RNase solution for 15 min (RNase I is a remarkably stable enzyme)
and cool to room temperature. Dispense into aliquots and store frozen.
As already mentioned (Section 2.2.), during the preparation of DNA,
endogenous DNases can be inhibited by the inclusion of EDTA in the
homogenization buffer. It was also pointed out that a DNase activity
had recently been discovered that was apparently stimulated by EDTA
(1). The prevalence of this type of nuclease activity is not known, but
if it is suspected, damage can be limited by performing extractions at
low temperatures and proceeding to the protein removal step as rap-
idly as possible.
3.4. Removal of RNase from DNase Solutions
RNase-free preparations of DNase I are now commercially avail-
able, however some of these still may not be entirely satisfactory.
Various column chromatography methods have been used to remove
RNase activity from DNase solutions (e.g., ref. 6) and an effective
method for disabling the RNase A enzyme has been described by
Zimmerman and Sadeen (7) and modified by Gurney and Gurney (8).
This method depends on the alkylation of a histidine in the active site
of RNase A (a likely contaminant of DNase I as they are derived from
the same tissue) using sodium iodoacetate. Abrief protocol is outlined
in the following:
- Use autoclaved micropipet tips and glassware.
- Dissolve 10 mg commercial DNase in 2 mL of 2.5 mM HC1.
- Dialyze the solution for a few hours at 40°C against 1 L of 2.5 mM HC1
with stirring, then overnight against 1 L of fresh 2.5 mM HCI again at
4°C with stirring. - Freshly prepare 1M sodium iodoacetate. Mix 2.5 mL of 0.2M sodium ace-
tate, pH 5.3, 2.0 mL of dialyzed enzyme, and 7.5 mL of 1M sodium iodo-
acetate. Incubate the mixture at 55°C for 60 min. A precipitate will form. - Dialyze the solution overnight against 1 L of 2.5 mM HCI at 4°C.
- Centrifuge solution at 10,000g for 30 min at 0°C.
- Carefully pipet off the supernatant into a sterilized tube or bottle with a
tight fitting cap. - The protein concentration should be 3-4 mg/mL. The DNase should be
stored at 4°C, the activity should be stable (unfrozen) for at least 2 yr.
RNases can also be inhibited by a number of compounds including
vanadyl ribonuclease complex, heparin, and their own specific ribo-