A potentially major source of contamination with ribonuclease
is the hands of investigators. Gloves should be worn at all stages
during the preparation of materials and solutions used for the
isolation and analysis of RNA and during all manipulations involving
Laboratory glassware is often a source of ribonuclease contamination
and should be treated by baking at 250oC for at least 6 hours. Glassware may be treated with a solution
of 0.1% diethylpyrocarbonate (DEPC), which is a strong but not
absolute inhibitor of RNase. Before using glassware treated with
a solution of 0.1% diethylpyrocarbonate, it is important to remove
traces of DEPC by heating to 100oC for 15 minutes or by autoclaving. Remaining traces of diethylpyrocarbonate
will inactivate the RNA by carboxymethylation.
Bake all glassware at 250oC for overnight.
Rinse all plasticware with 0.1 N NaOH, 1 mM EDTA followed by a solution of 0.1% diethylpyrocarbonate (DEPC).
Treat all solutions with 0.05% DEPC for 12 hours at 37oC, and then autoclave for 30 minutes.
Tris buffer can not be treated with diethylpyrocarbonate. DEPC
is highly unstable in the presence of Tris buffer and decomposes
rapidly into ethanol and CO2.
Fedorcsak, I, Ehrenberg, L (1966) Effects of diethylpyrocarbonate
and methyl methanesulfonate on nucleic acids and nucleases. Acta
Chem. Scand. 20: 107.
Kumar, A, Lindberg, U (1972) Characterization of messenger
ribonucleoprotein and messenger RNA from KB cells. PNAS 69: