EDTA (Ethylenediaminetetraacetic acid), a polyamino carboxylic acid, is extensively used in molecular biology experiments as a chelating agent. It efficiently sequesters divalent metal ions that are required for many nucleic acid modifying enzymes including DNases (but not RNases). EDTA-sequestered metal ions, although present in the solution (no precipitation), are unable to catalyze any enzymatic reactions. This property of EDTA makes it a suitable choice for molecular biologists to stop an enzymatic reaction or protect nucleic acid from any unwanted modification or degradation.
EDTA is commercially available in several forms. Disodium salt of EDTA (EDTA.Na2.2H2O) is most commonly used in molecular biology. A 0.5 M EDTA solution can be prepared by dissolving 186.12 grams of EDTA.Na2.2H2O (Molecular Weight: 372.24) in water to a final volume of 1000 ml.
Reagents and solutions
NaOH pallet / 10N NaOH solution (for pH adjustment)
Deionized / Milli-Q water
Equipment and disposables
Conical flask / Beaker
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 a 2-liter beaker / conical flask. Add 800 ml deionized / Milli-Q water.
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: When all EDTA is completely dissolved, adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.
Step 4: Transfer the solution to an autoclavable bottle and sterilize it by autoclaving for 20 minutes at 15 lb/in2 (1.05 kg/cm2) on liquid cycle.
One can sterilize the solution by filtering through a 0.22 μm filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μm which includes most bacteria and their spores but not mycoplasma. In addition, it does not inactivate enzymes (e.g., DNases). In contrast, autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.
The solution can be stored at 15 – 25 °C (room temperature) for years.
|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|
Calculate the amount of EDTA.Na2.2H2O (M.W 372.24 g/mol) required to prepare a solution of specific molarity and volume.
Molecular weight of EDTA.Na2.2H2O: 372.24 g/mol
Molarity of EDTA solution: M
(Change the molarity of the solution)
Volume of stock solution: ml
(Change the volume of the solution)
Required amount of EDTA.Na2.2H2O: 186.12 g