CaEGTA Solution (100 mm)

• 1. Add 1.001 g of CaCO₃ in a glass beaker.

• 2. Add 3.804 g of EGTA (e.g., Sigma-Aldrich >99%) and 50 mL ultrapure water.

• 3. Add 20 mL of 1 m KOH (make fresh from KOH concentrate, e.g., Fluka). (The solution will effervesce after addition of KOH.)

• 4. Add 0.238 g HEPES free-acid form (e.g., Sigma-Aldrich ≥99.5%; 25 mm final concentration).

• 5. Stir the solution. Heat to ∼70°C to allow all the CaCO₃ to dissolve (can take >20 min).

• 6. Add KOH until pH is ∼7.0.

• 7. Cool to room temperature and add more KOH until the pH is 7.0.

• 8. Store in plastic bottles at 4°C until use. (The solution is good for several months.)

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