DEPC water vs autoclaved water is a question that only really has one right answer once you know what each process actually does to RNase, and there’s a persistent lab myth on both sides of it worth clearing up first.
Use DEPC-treated water for RNA-sensitive work, where reliable RNase inactivation matters. Standard autoclaved water is fine for general molecular biology that isn’t RNA-sensitive.
What DEPC Actually Does
Diethyl pyrocarbonate (DEPC) inactivates RNase by covalently modifying the enzyme’s histidine residues, along with lysine, cysteine, and tyrosine to a lesser extent. That chemical modification destroys the enzyme’s activity directly, rather than relying on heat to denature it. Water is typically treated with 0.1% DEPC for at least two hours, then autoclaved to break the DEPC down into carbon dioxide and ethanol, both of which are harmless byproducts. That final autoclave step isn’t optional: DEPC itself is a reactive, toxic compound, and it has to be hydrolyzed away before the water is usable, at a DEPC concentration of 0.1%, roughly 15 minutes of autoclaving per liter is enough to leave the water DEPC-free.
The Myth About Autoclaving Alone
There’s a common belief in cell culture and molecular biology labs that plain autoclaving does nothing at all to RNase, and that belief isn’t quite accurate. Autoclaving does inactivate a meaningful amount of RNase A, RNases are heat-tolerant compared to most enzymes, not heat-proof. The real issue is reliability: autoclaving alone doesn’t consistently or completely eliminate RNase activity the way DEPC’s covalent chemical modification does, and RNases that survive a heat cycle can sometimes refold and regain partial activity afterward. For RNA work, where a trace of surviving RNase can degrade your sample before you ever get to run it, that unreliability is the whole problem.
When Each One Is the Right Call
- DEPC-treated water: use for RNA extraction, RNA storage, RT-PCR, and any application where residual RNase activity, even a small amount, would compromise your results.
- Standard autoclaved water: perfectly adequate for general molecular biology work that isn’t RNA-sensitive, DNA work, general buffer preparation, and routine lab use where RNase contamination isn’t a meaningful risk.
Using DEPC water everywhere isn’t wrong, but it is unnecessary cost and prep time for work that was never at risk from RNase in the first place. The decision should track the actual sensitivity of what you’re doing, not a blanket policy.
PurMa Biologics manufactures Ultra-Pure DEPC Treated Nuclease-Free Water for RNA-sensitive applications, Non-DEPC Nuclease-Free Water for general nuclease-sensitive work, and General Molecular Biology Grade Water for routine applications. See our product pages for full specifications.
