TL;DR: To make 1 L of 0.5 M EDTA using disodium EDTA dihydrate, weigh 186.12 g, suspend in about 800 mL water, then raise the pH gradually with NaOH. EDTA will not fully dissolve until the pH approaches 8.0. Once dissolved and at pH 8.0, bring to 1 L.
EDTA is a strong chelator of divalent metal ions and is a core stock solution in molecular biology. A 0.5 M EDTA stock is commonly used to make TE, TAE, TBE and other nucleic-acid buffers.
0.5 M EDTA recipe
The masses below assume disodium EDTA dihydrate, molecular weight 372.24 g/mol.
| Final volume | Disodium EDTA dihydrate |
|---|---|
| 100 mL | 18.61 g |
| 500 mL | 93.06 g |
| 1 L | 186.12 g |
Why EDTA initially refuses to dissolve
This is normal. At low pH, EDTA is poorly soluble. As NaOH is added and the solution approaches pH 8, the EDTA becomes more ionised and dissolves. Do not keep adding water simply because solid remains.
How to make 1 L of 0.5 M EDTA pH 8.0
- Add approximately 800 mL purified water to a large beaker with strong stirring.
- Add 186.12 g disodium EDTA dihydrate. Expect a suspension.
- Add NaOH gradually while monitoring pH. The solution will clear as the pH approaches 8.0.
- Once fully dissolved, adjust precisely to pH 8.0.
- Bring to 1 L with purified water.
- Sterilise if required by the downstream workflow.
Safety
Sodium hydroxide is strongly caustic and dissolution is exothermic. Add NaOH slowly with appropriate eye/skin protection. If using concentrated NaOH solution, handle it as a corrosive reagent.
What EDTA does in molecular biology
Many nucleases require Mg²⁺ or other divalent ions. EDTA chelates these ions and can reduce nuclease activity. Primary studies have shown improved preservation of high-molecular-weight DNA under selected EDTA-containing conditions.
When EDTA is a problem
EDTA can inhibit any downstream enzyme that requires divalent cations. That includes many polymerases, ligases, restriction enzymes and metalloproteins. The effect depends on how much EDTA enters the final reaction relative to Mg²⁺.
Common uses of 0.5 M EDTA stock
- TE buffer
- TAE and TBE electrophoresis buffers
- DNA/RNA preservation buffers
- Selected lysis and extraction buffers
- Stopping or slowing metal-dependent enzyme reactions
Troubleshooting
| Problem | Cause | Fix |
|---|---|---|
| EDTA will not dissolve | pH still too low | Continue gradual NaOH addition while stirring. |
| pH overshoots | NaOH added too quickly | Approach pH 8 slowly using smaller additions. |
| Downstream PCR is inhibited | Excess EDTA chelating Mg²⁺ | Dilute/clean DNA or use lower-EDTA storage buffer. |
Frequently asked questions
Can I make EDTA stock from free-acid EDTA?
Yes, but the required mass and NaOH demand are different. This recipe is specifically for disodium EDTA dihydrate.
Why is pH 8 commonly used?
EDTA is more soluble and more highly ionised near pH 8, improving metal chelation and making the stock easier to use reproducibly.