Diethyl pyrocarbonate (DEPC)-treated water is a specially prepared reagent used in molecular biology laboratories where the stability of Ribonucleic Acid (RNA) is paramount. RNA is highly susceptible to degradation by Ribonucleases (RNases), a class of enzymes that are exceptionally stable and ubiquitous, existing on surfaces, in dust, and even on human skin. The presence of these enzymes, even in minute amounts, can quickly destroy an RNA sample, rendering an experiment useless. This specialized water is necessary for any procedure involving the isolation, manipulation, or analysis of RNA, such as Northern blotting or in vitro transcription. The preparation process uses the chemical diethyl pyrocarbonate to inactivate lurking RNases before the water is used to prepare sensitive solutions and reagents.
Required Equipment and Initial Water Quality
Preparation begins with selecting the correct starting materials. The source water must be of extremely high quality, typically deionized, double-distilled, or reverse osmosis-purified water, often referred to as molecular biology grade water. Using water of lesser purity is not an option, as it may contain contaminants that could interfere with the subsequent chemical treatment or RNA stability.
The containers used must be heat-resistant, such as an autoclavable glass bottle, and dedicated exclusively to RNA work to prevent cross-contamination. Glassware should be rendered RNase-free before use by baking it in a high-temperature oven, such as at 240°C for at least four hours. This baking step destroys any existing RNase contamination on the glass surface. The final prerequisite is the chemical agent itself, Diethyl pyrocarbonate, which is a clear, colorless liquid.
The Chemical Treatment Phase
Once the high-quality water is measured into the cleaned glassware, the chemical inactivation process begins with the addition of DEPC. The standard concentration used is 0.1% volume-to-volume (v/v), translating to 1 milliliter of DEPC for every 1 liter of water. Since DEPC has limited solubility, it will initially appear as small globules upon addition.
The mixture must be vigorously shaken or stirred until the DEPC globules completely disappear and the solution appears clear. This ensures the chemical is properly dispersed throughout the water to interact with all potential RNase molecules. Following mixing, the solution is incubated for a specific period, typically for at least 12 hours at 37°C, or alternatively, overnight at room temperature.
During this incubation, DEPC acts as a potent nuclease inhibitor, irreversibly reacting with and modifying the active sites of RNase enzymes. DEPC reacts most strongly with histidine residues within the enzyme’s structure, but it can also modify lysine, cysteine, and tyrosine residues. This covalent modification permanently inactivates the RNases, preventing them from degrading RNA in downstream applications.
Inactivation and Removal of DEPC
Following RNase inactivation, the next step is to completely remove any residual DEPC from the water. Remaining traces of DEPC are undesirable because the chemical can modify purine residues in RNA, a process called carboxymethylation. This modification can inhibit subsequent enzymatic reactions, such as in vitro transcription.
The standard method for removing DEPC is through autoclaving, which involves heating the solution to 121°C under pressure. Prior to autoclaving, the cap on the glass bottle must be loosened slightly to prevent pressure buildup from the gases released during the process. The high heat and pressure accelerate the hydrolysis of DEPC, causing it to decompose into two harmless byproducts: ethanol and carbon dioxide gas.
For a 0.1% DEPC solution, autoclaving for at least 15 minutes per liter of water is sufficient to break down all traces of the chemical. Once the autoclaving cycle is complete, the water is both RNase-free and DEPC-free. If an autoclave is unavailable, an alternative method involves heating the solution to 100°C for 15 minutes, which can be accomplished by boiling the solution.
Safe Handling and Water Storage
Because concentrated DEPC is a hazardous chemical and a suspected carcinogen, safety precautions must be followed when handling the liquid. The chemical should always be handled in a certified chemical fume hood to manage the vapors. Users must wear appropriate personal protective equipment, including a lab coat, eye protection, and chemical-resistant gloves. DEPC is sensitive to moisture and can decompose to produce carbon dioxide, which may cause pressure to build up in the stock bottle over time.
Once the DEPC-treated water is prepared and cooled, it should be stored in small, tightly sealed aliquots in new, sterile, or baked containers. Storing the water in smaller volumes minimizes the risk of contaminating the entire batch if one aliquot is exposed to airborne RNases or non-sterile equipment. Aliquots suspected of contamination, such as those opened repeatedly, should be discarded immediately to maintain the integrity of future RNA experiments.