Preparation of 0.5 M EDTA solution

Preparation of 0.5 M EDTA solution from Disodium ethylenediaminetetraacetate dihydrate (EDTA.Na2.2H2O)


  • EDTA (Ethylenediaminetetraacetic acid) is a polyamino carboxylic acid. It is extensively used in molecular biology experiments as a chelating agent.
  • It sequesters metal ions such as Ca2+ and Fe3+. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity.
  • Metal ions are necessary for the action of many enzymes including DNases.
  • EDTA is commercially available in several forms. Disodium salt of EDTA is most commonly used in molecular biology.
  • 0.5 M EDTA solution is used for the preparation of many solutions including TAE, TBE, DNA loading dye, resuspension buffer (isolation of plasmid), Tris-EDTA, Trypsin-EDTA, etc.

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  • Reagents
    •  EDTA.Na2.2H2O
    • NaOH pallet / 10N NaOH solution (for pH adjustment)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer


  • 0.5 M EDTA, pH 8.0 at 25°C


Preparation of 1000 ml of 0.5 M EDTA solution, pH 8.0 in water from ethylenediaminetetraacetic acid disodium salt, dihydrate (EDTA.Na2.2H2O)


Step 1: Weigh out 186.12 grams of EDTA.Na2.2H2O (Molecular Weight 372.24). Transfer to 2 L beaker / conical flask. Add 800 ml deionized / Milli-Q water.

Precaution: Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.

Step 2: While stirring vigorously on a magnetic stirrer, add NaOH pellet or 10N NaOH to adjust the solution pH 8.0.

  • ~20 g NaOH pellet is required to adjust the pH 8.0.
  • It is not easy to dissolve EDTA. To dissolve the EDTA completely, solution pH 8.0 is required.

Step 3:  Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.

Step 4:  Transfer the solution to autoclavable bottle. Sterilize the solution by autoclaving (20 minutes at 15 lb/ (psi) from 121-124°C on liquid cycle)

  • Depending on the consumption, one can make small aliquots of solution.
  • One can sterilize the solution by passing through 0.22 μ filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μ which includes most bacteria their spores but not mycoplasma. However, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.


Solution can be stored at 15 – 25 °C (room temperature) for several months.

Follow the table to prepare EDTA solution of specific concentration and volume from EDTA.Na2.2H2O (Molecular Weight 372.24).
Conc. / Volume 100 ml 250 ml 500 ml 1000 ml
10 mM 0.37 g 0.93 g 1.86 g 3.72 g
100 mM 3.72 g 9.30 g 18.61 g 37.22 g
0.25 M 9.31 g 23.26 g 46.53 g 93.06 g
0.5 M 18.61 g 46.53 g 93.06 g 186.12 g