Hi,
I need to prepare the following elution buffer:
- 40mM Tris/HCL, pH 8
- 10mM EDTA
- 3.5M Urea
- 0.02% Tween
I had previously made this buffer by calculating the mass of each ingredient so for a 500mL flask 2.428g Tris, 1.871g EDTA and 105.1g Urea. I mixed everything together in 400-480ml dH2O and then adjusted the pH of the solution to 8.
I now tried to prepare a new one but after adding all the ingredients together the pH was already well below 8...
I can't figure out what the problem might be. Anyone has any ideas why this might be happening?
likely the first time you used Trizma as Tris. And the 2nd time you used Trizma hydrochloride.
Hi there,
As mentioned aboveTris might be the issue but also EDTA might be (make sure you use the neutralized acid form, ie di or tri sodium salt)
I also used standard solutions to check the accuracy of the pH meter and it didn't need any calibrating
Just a general update. The problem was the EDTA. The first time I had used disodium EDTA whereas the second time I used plain EDTA, which has a significantly different pH.
Moral of the story: Make sure almost identical bottles of a similar compound are easily distinguishable in the lab
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