Molecular Techniques and Methods

Removal of RNase Contamination
from Materials and Solutions

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

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 RNA.

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.




PROCEDURES

  • 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.





  • REFERENCES

  • 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: 681.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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