How to Make DEPC Water for RNase-Free Experiments

Making DEPC-treated water involves adding a small amount of diethyl pyrocarbonate to purified water, letting it react long enough to destroy any RNase enzymes present, and then autoclaving the water to break down the DEPC itself before use. The standard working concentration is 0.05% DEPC by volume, and the entire process takes a few hours of incubation followed by a standard autoclave cycle.¹ It sounds simple, and mechanically it is, but each step has pitfalls that can ruin RNA work downstream if you cut corners.

Why DEPC Water Exists

RNases are among the most stubborn enzymes in molecular biology. They are remarkably stable, survive boiling and brief autoclaving, and exist on skin, glassware, and reagent surfaces in quantities sufficient to degrade RNA within seconds. Standard distilled or deionized water, even high-quality water, can harbor enough RNase contamination to compromise sensitive RNA experiments. DEPC solves this by chemically attacking RNases at the molecular level, permanently disabling them.

DEPC works by modifying histidine residues in the active site of RNase enzymes. Specifically, it carbethoxylates the imidazole rings of critical histidine residues, which are essential for the enzyme’s catalytic activity.² Once those residues are chemically blocked, the enzyme can no longer cleave RNA. This is an irreversible inactivation, not just a temporary inhibition. The RNase is permanently destroyed.

Step-by-Step Preparation

The procedure itself is straightforward, but attention to detail matters at every stage. Here is how to do it properly:

• Start with good water: Use distilled or deionized water as your base. The cleaner your starting water, the less interference you will encounter later. Tap water or low-grade deionized water introduces mineral and organic contaminants that can react with DEPC or interfere with downstream reactions.
• Add DEPC to 0.05% (v/v): For one liter of water, add 0.5 mL of liquid DEPC. Use a glass or chemical-resistant pipette, not a plastic one that DEPC might degrade. Work in a fume hood because DEPC vapor is irritating and the chemical is a suspected carcinogen.
• Mix thoroughly: Shake the bottle vigorously on an orbital platform for several hours, or stir with a magnetic stirrer for 20 to 30 minutes.¹ DEPC is not very soluble in water, so inadequate mixing means parts of your solution never actually encounter enough DEPC to destroy RNases. You will often see oily droplets floating on the surface if you have not mixed long enough.
• Autoclave to destroy the DEPC: After incubation, autoclave the treated water on a standard liquid cycle (typically 121 °C, 15 psi, for 15 to 20 minutes). At autoclaving temperature and pressure, DEPC breaks down into carbon dioxide and ethanol, both of which are volatile and escape during the cycle.¹
• Cool and store: Let the water cool to room temperature, then store it in RNase-free containers with tight caps. Aliquoting into smaller volumes reduces the number of times you open a single bottle and risk recontamination.

The whole process from adding DEPC to having usable water takes roughly half a day when you account for mixing time and the autoclave cycle. Many labs prepare several liters at once so they have a ready supply.

Why the Autoclave Step Is Not Optional

Some researchers, especially those new to RNA work, treat the autoclaving step as a formality. It is not. DEPC that remains in your water after treatment will react with the very RNA you are trying to protect. Even trace amounts of residual DEPC chemically modify adenine bases in RNA, which changes the molecule’s physical properties and compromises its use in translation assays, blot analysis, and PCR-based applications.³ In other words, un-autoclaved DEPC water can do more damage to your RNA than the RNases it was supposed to eliminate.

The chemistry here is worth understanding in broad strokes. During autoclaving, DEPC undergoes hydrolysis and breaks apart into ethanol and CO₂.⁴ Both are volatile under autoclave conditions and mostly evaporate away. However, small amounts of ethanol can remain and react with impurities or reagents in later experimental steps. For most RNA applications this residual ethanol is harmless at the concentrations present, but it is one of the reasons some labs have moved toward alternative approaches for obtaining RNase-free water.

Concentration Matters More Than You Think

The standard recommendation of 0.05% DEPC is not arbitrary. Using too little means some RNases survive. Using too much creates more residual DEPC that the autoclave step has to eliminate, and it increases the chance that some reactive DEPC survives autoclaving. You will occasionally see protocols calling for 0.1% DEPC, which is twice the standard concentration. This can work, but it requires more thorough autoclaving and carries a higher risk of leaving behind active DEPC.

The mixing step also directly affects the effective concentration. Because DEPC is poorly soluble in water, insufficient mixing means the DEPC stays concentrated in droplets rather than distributing evenly. The water near those droplets gets over-treated while the rest of the bottle never sees enough DEPC to inactivate RNases. If you shake by hand for 30 seconds and call it done, you have not made DEPC water. You have made water with some oily blobs of DEPC floating in it.

What DEPC Water Should and Should Not Be Used For

DEPC-treated water is appropriate for preparing buffers and solutions used in RNA extraction, Northern blotting, in situ hybridization, and reverse transcription reactions. It is also used for rinsing glassware and equipment that will contact RNA. Some microfluidic applications use DEPC-treated water as part of probe-washing protocols to maintain an RNase-free environment during single-cell RNA assays.⁵

There are situations where DEPC water should not be used. Never use it to prepare Tris-based buffers. DEPC reacts with the primary amine group in Tris, which neutralizes the DEPC before it can inactivate RNases. If you need an RNase-free Tris buffer, prepare the Tris solution using water that was DEPC-treated and autoclaved first, then add the Tris powder to the already-treated water. The same caution applies to other buffers containing primary amines, such as HEPES.

DEPC water is also unnecessary for DNA work. DNA is far more resistant to nuclease degradation than RNA, and standard autoclaved water or commercial nuclease-free water is typically sufficient for DNA applications.

Alternatives to DEPC Treatment

DEPC treatment has been the gold standard for decades, but it is not the only way to obtain RNase-free water. Several alternatives have gained traction, particularly in labs that find the DEPC protocol cumbersome or want to avoid handling a suspected carcinogen.

Commercial nuclease-free water is the most common alternative. These products are manufactured under stringent conditions and tested to ensure they are free of RNase and DNase activity. They cost more per liter than making your own DEPC water, but they eliminate the preparation time and the risk of user error during DEPC treatment. For labs that use relatively small volumes, the convenience often outweighs the cost.

Advanced water purification systems offer another route. Research comparing ultrapure water from dedicated purification systems with DEPC-treated water found no significant difference in RNA degradation between the two.⁶ Hybridization experiments with RNA transcripts of different sizes showed that the ultrapure water performed just as well for RNA preparation and Northern blot analysis. Separately, studies comparing ultrafiltration-based water purification with DEPC treatment confirmed that ultrafiltration is equally efficient at suppressing RNase activity.⁷ The practical takeaway is that if your lab already has a high-end water purification system with ultrafiltration capabilities, you may not need DEPC treatment at all.

That said, many labs stick with DEPC-treated water because they trust the chemical inactivation step as a definitive kill of any RNases present, rather than relying on a physical filtration step that might allow occasional breakthrough. The choice often comes down to lab culture, budget, and how critical your RNA work is. For high-stakes experiments like RNA-seq library preparation or quantitative RT-PCR, some researchers use both approaches in sequence: starting with ultrapure water and then DEPC-treating it for an extra layer of assurance.

Safety and Handling Precautions

DEPC is classified as a suspected carcinogen and is an irritant to the eyes, skin, and respiratory tract. Always work with it in a chemical fume hood. Wear gloves (nitrile is preferred) and safety glasses. Avoid breathing the vapor. If DEPC contacts your skin, wash the area immediately with soap and water.

The liquid is stored at 4 °C (in a refrigerator, not a freezer) and has a limited shelf life once opened. Exposure to moisture causes DEPC to slowly hydrolyze in the bottle, so keep the cap tightly sealed and check the expiration date. Old DEPC that has partially degraded will be less effective at inactivating RNases, which means your treated water might not actually be RNase-free even if you followed the protocol correctly. If the DEPC smells different than it did when fresh, or if you see signs of degradation in the bottle, replace it.

After autoclaving, the DEPC in your treated water has broken down and the water itself is safe to handle without special precautions. The hazard is only during the pre-autoclave mixing step when active DEPC is present.

Common Mistakes That Compromise DEPC Water

Even experienced researchers occasionally produce DEPC water that fails to protect RNA. The most frequent errors fall into a few categories:

• Insufficient mixing: As discussed above, DEPC does not dissolve easily. A quick hand-shake is not enough. Use a stir plate or orbital shaker for the full recommended duration.
• Skipping or shortening the autoclave cycle: A partial autoclave run leaves active DEPC in the water, which then damages RNA. Run a full liquid cycle every time.
• Treating Tris buffers directly: DEPC reacts with primary amines and gets inactivated. Prepare DEPC water first, autoclave it, and then dissolve Tris or other amine-containing reagents.
• Contaminating after preparation: Opening the bottle repeatedly, using non-sterile pipette tips, or pouring DEPC water into dirty containers reintroduces RNases. Aliquot into smaller volumes and use dedicated RNase-free plasticware.
• Using degraded DEPC: An old bottle of DEPC that has been sitting open at room temperature may have lost much of its potency. Always store DEPC properly and replace it when it is past its expiration.

Verifying That Your DEPC Water Actually Works

Making DEPC water is only useful if you confirm it is doing its job. The simplest quality-control check is to run a known intact RNA sample (total RNA from cells, for example) in your DEPC-treated water alongside a positive control in commercial nuclease-free water. If the RNA in your DEPC water shows the same sharp ribosomal RNA bands on an agarose gel, your water is good. Degraded RNA appears as a smear rather than distinct bands.

For more rigorous testing, some labs use fluorescent RNase activity assays. These assays use an RNA substrate labeled with a fluorescent dye and a quencher; when RNase cleaves the substrate, the fluorescence increases. Such assays are sensitive enough to detect low-level RNase contamination that a gel might miss.⁸ These are more relevant for clinical or diagnostic settings where even trace RNase activity could invalidate results, but they are available from several commercial suppliers for any lab that wants the extra certainty.

A practical note: if your RNA experiments are consistently producing degraded results and you suspect the water, test the water separately from other reagents. RNase contamination can also come from pipette tips, tubes, gel-casting equipment, or the researcher’s own hands. DEPC water fixes only one source of contamination. Good RNase-free technique extends to every surface and reagent in the workflow.

DEPC Treatment for Glassware and Plasticware

DEPC water is not only used as a reagent solvent. Many labs also use it to rinse glassware, gel combs, and electrophoresis tanks before RNA experiments. The idea is the same: flood the surface with DEPC-containing water, let it sit long enough to inactivate any adsorbed RNases, and then autoclave or heat-treat the item to destroy residual DEPC.

For glassware, an alternative is to bake items at 180 °C for four hours or more. This dry-heat sterilization destroys RNases by thermal denaturation and avoids DEPC entirely. Baking works well for glass but will melt or deform most plastics, so it is limited to heat-resistant materials. Plastic consumables (tubes, tips) are better purchased as certified RNase-free from the manufacturer, or treated with a brief DEPC water rinse followed by thorough drying.

One often-overlooked source of contamination is the autoclave itself. If the autoclave is used for general lab sterilization, its chamber and steam lines may harbor trace amounts of biological material including RNases. Some RNA-focused labs dedicate an autoclave cycle to RNase-free work only, or at least run an empty sterilization cycle before autoclaving their DEPC-treated water and glassware.

When DEPC Water Is Overkill

Not every molecular biology experiment needs DEPC-treated water. If you are working with DNA, proteins, or small molecules, standard autoclaved water or commercial molecular-biology-grade water is perfectly adequate. Even within RNA work, some applications are more forgiving than others. A quick diagnostic RT-PCR from a high-abundance target can often tolerate minor RNase contamination that would destroy a low-input RNA-seq library.

The trend in recent years has been toward commercially prepared nuclease-free water for routine RNA work, reserving DEPC treatment for situations where the lab needs large volumes of RNase-free water at low cost or where institutional protocols require it. High-throughput genomics facilities, for instance, tend to buy certified nuclease-free water in bulk rather than dedicating staff time to DEPC preparation. Smaller academic labs with tighter budgets often find that making their own DEPC water is the more economical choice, especially if they already have DEPC on hand and an autoclave available.

The science behind making DEPC water has not changed in decades. What has changed is the availability of alternatives and the increasing sensitivity of downstream applications. Whether you make your own or buy it, the goal is the same: water that will not destroy your RNA before you get a chance to study it.