DIETHYL PYROCARBONATE (DEPC) TREATMENT OF
AQUEOUS SOLUTIONS

Diethyl pyrocarbonate derivitizes histidine residues and is therefore an effective method to inactivate nucleases including RNAse. CAUTION: DEPC is irritating to the eyes, skin, and mucous membranes. It is a suspected carcinogen. It hydrolitically decomposes to CO2 & ethanol and may decompose to urethane (a possible carcinogen) in the presence of ammonia. Use it in the fume hood and wear gloves! DEPC is highly unstable in Tris buffers so avoid use of TE for DNA dilutions prior to DEPC treatment whenever possible. Th e CO2 generated during the decomposition of DEPC may reduce the pH of the solution. It is therefore best to EtOH precipitate the DNA and redissolve it in the appropriate buffer before further manipulations are undertaken.
  1. Use DEPC only from a recently opened container (one month old or less). Be sure to wear gloves when working with the DEPC, and open it only in the fume hood. Due to build up of pressure or faulty glass, Sigma has repor ted bottles of DEPC have exploded!

  2. Dilute the DEPC 1:100 in absolute ethanol.

  3. For 1X DEPC treatment, add 1/10 volume of the 1:100 diluted DEPC (from step 2) to 1 volume of solution to be treated. Thus, the working concentration of DEPC is 0.1% v/v. For DNA treatment, the total volume should be at least 100µl to avoid evaporation in the next step. Plasmid preps may need a 3-4x DEPC treatment (0.3 - 0.4% by volume).

  4. Vortex and incubate at 65°C for o ne hour WITH THE CAP OFF THE MICROFUGE TUBE to release the CO2 that will be generated. The reaction is complete when bubbles stop forming in the tube. For treatment of large aqueous volumes, incubate at 37-42°C overnight followed by 45 minutes of autoclaving. The DEPC smell should be gone.

    Send comments and updates to  D r. Bart Frank, Arthritis and Immunology Program, OMRF
Return to Protocols: Table of Contents
Return to the Frank Lab Home Page
Original document prepared by Renee M. Besta, May 1984
Copyright 1993, 1996, and 1997 by Mark Barton Frank, P h.D.
Proper citation for data acquired from this document is: "Besta, R. M. and Frank, M. B. Diethyl Pyrocarbonate (DEPC) Treatment of Aqueous Solutions. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/depc.html). 1997. Oklahoma City. Revision Date: October 2, 1997."