( d by adding 1 ml DEPC per liter ...)Decontamination and inhibition of ribonucleases

d by adding 1 ml DEPC per liter of solution, stirring for 1 hour, and autoclaving for one hour to hydrolyze any remaining DEPC into CO2 and Ethanol. Compounds with primary amine groups (e.g. Tris) will react with DEPC. Consequently, Tris buffers should be prepared by dissolving Tris base (from fresh bottle reserved for RNA work) in DEPC-treated and autoclaved water, adjusting the pH (with an electrode reserved for RNA work), and re-autoclaving to sterilize. Solutions of thermolabile materials (e.g. DTT, nucleotides, manganese salts) should be prepared by dissolving the solid (highest available purity) in DEPC-treated and autoclaved water and passing the solution through a 0.2 m filter to sterilize2., 3.. As an alternative to DEPC, which inhibits enzymatic reactions and modifies both RNA and Tris if not completely hydrolyzed, we recommend the use of RNase-free Molecular Biology Grade Water.

Use of ribonucleases inhibitors
A very convenient and effective way to protect RNA from decomposing RNase activity is the use of an RNase inhibitor. Eppendorf Prime RNase Inhibitor is a protein of non-human origin that binds non-covalently to RNase and inhibits the same type of ribonucleases as human placental RNase inhibitors (HPRI), including RNases A, B, and C. Prime RNase Inhibitor is stable under a broad range of pH, DTT concentrations and temperatures. It inhibits greater than 90% of RNase activity in reactions where an equal amount of HPRI inhibits only 50%.

References:
1. Blackburn, P., and Moore S. (1982) The Enzymes. Academic Press, NY, 317.
2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press,
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