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Short answer
DEPC (diethyl pyrocarbonate) is a chemical added to water, at 0.1% in Thermo Fisher’s (Ambion) guidance, to inactivate RNases before the water is used for RNA work. It is then removed by autoclaving. It cannot be used in every solution: Tris and HEPES buffers use it up, and leftover DEPC can suppress enzymatic reactions or chemically alter RNA.
Definition and scope
DEPC is an RNase-inactivating additive used when preparing water and some solutions for RNA experiments. Thermo Fisher’s Ambion technical note states that 0.1% DEPC “is probably adequate to inactivate most RNase contamination from environmental sources.” The same note is clear that this is not unlimited: the amount of DEPC needed rises with the amount of contaminating RNase. In their test 0.01% DEPC was insufficient at 500 ng/ml RNase A, and increasing the concentration to 0.1% protected the probe up to that level. This page covers what DEPC does and which solutions it suits. For the wider extraction workflow, see RNA extraction protocol basics.
How DEPC treatment works in practice
Bitesize Bio explains that DEPC reacts most strongly with histidine residues but can also react with lysine, cysteine and tyrosine residues, and that RNases usually rely on histidine, tyrosine or serine as a nucleophile, which is why modification by DEPC disables them.
Thermo Fisher’s product FAQ for DEPC-treated water gives the preparation as 1 mL of DEPC per litre of water, mixed until dissolved, left to stand for one hour, then autoclaved. The Ambion note’s test protocol is similar: shake vigorously for one minute, incubate for one hour at room temperature, then autoclave for 25 minutes. Bitesize Bio gives 37 °C for 2 hours, or room temperature overnight, as alternative incubations.
The autoclave step matters as much as the incubation. Per the Ambion note, DEPC has a half-life of roughly 30 minutes in water, and at 0.1% a solution autoclaved for 15 minutes per litre can be assumed DEPC-free. Bitesize Bio notes that autoclaving breaks DEPC down into carbon dioxide, ethanol and water. For how autoclaves are used and validated in labs, see what is an autoclave.
Which solutions can and cannot be DEPC-treated
This is the part most summaries leave out. The Ambion note reports that Tris contains an amino group that “sops up” DEPC and makes it unavailable to inactivate RNase. Tris and HEPES made DEPC unavailable at 0.1%; MOPS and PBS did not show this effect. Overcoming Tris/HEPES interference required 1% DEPC. Thermo Fisher’s product FAQ states plainly that buffers containing Tris cannot be DEPC-treated.
| Solution | What the Thermo Fisher sources report |
|---|---|
| Water | Treated with DEPC, then autoclaved (the product FAQ and Ambion note describe this). |
| Tris buffers | Cannot be DEPC-treated: Tris binds DEPC through its amino group. Order given in the FAQ: treat and autoclave the water, add the Tris, then autoclave again. |
| HEPES buffers | Made DEPC unavailable at 0.1% in the Ambion test; 1% was needed to overcome the interference. |
| MOPS and PBS | Did not show the interference effect in the Ambion test. |
For the most cautious route to RNase-free buffers, the Ambion note recommends mixing molecular-biology-grade powdered reagents in DEPC-treated water. General buffer preparation is covered in buffer and solution preparation.
Residual DEPC and downstream enzymes
The Ambion note warns that high levels of residual DEPC, or its by-products, in a solution can inhibit some enzymatic reactions or chemically alter (carboxymethylate) RNA. It quantifies the effect on transcription: incorporation fell as DEPC concentration rose, to 64% at 0.01%, 59% at 0.10% and 53% at 1.00% DEPC. That is the trade-off in one line: higher DEPC doses inactivate more RNase, but leave more to remove before enzymatic steps such as in vitro transcription. Assays that begin from RNA, such as RT-qPCR, depend on the RNA staying intact, which can be checked with a metric like the RNA Integrity Number.
DEPC-treated water, autoclaved water and nuclease-free water
For anyone deciding what water to stock or make, Thermo Fisher’s nuclease-free water page defines nuclease-free water as “water that has been deionized, filtered, autoclaved, and tested for contaminants.” It states that autoclaving alone may be sufficient to inactivate a substantial amount of RNase enzyme, and that if downstream assays are sensitive, DEPC-treated nuclease-free water is a better option. The choice therefore follows how sensitive the downstream assay is, not a single rule.
Common misunderstandings
“DEPC works in any buffer”
No. Tris and HEPES use up DEPC at 0.1% (see the table above).
“DEPC water is DEPC-free once it is made”
Only after the autoclave step. Per the Ambion note, at 0.1% a solution autoclaved for 15 minutes per litre can be assumed DEPC-free; an unautoclaved treated solution still carries DEPC.
“0.1% handles any amount of RNase”
Not always. The note describes 0.1% as adequate for most environmental contamination, and the DEPC needed rises with the RNase load: 0.01% was insufficient at 500 ng/ml RNase A, while 0.1% protected the probe up to that level.
FAQ
What does DEPC stand for?
Diethyl pyrocarbonate. It is added to water or solutions to inactivate RNases for RNA work.
What concentration of DEPC is used?
0.1% (1 mL per litre) in the Thermo Fisher sources, followed by incubation and autoclaving.
Can I add DEPC to Tris buffer?
No. Thermo Fisher states that buffers containing Tris cannot be DEPC-treated. Prepare DEPC-treated, autoclaved water first, then dissolve the Tris and autoclave again.
Sources
- Thermo Fisher Scientific (Ambion), RNase and DEPC treatment, technical note, checked 2026-10-29.
- Thermo Fisher Scientific, DEPC-Treated Water (4387937) FAQs, checked 2026-10-29.
- Thermo Fisher Scientific, Nuclease-free water, checked 2026-10-29.
- Bitesize Bio, DEPC: The Wicked Witch of RNA?, used for mechanism and incubation alternatives, checked 2026-10-29.
