TPE is a commonly used buffer for DNA electrophoresis. This buffer contains 89 m
ID: 784440 • Letter: T
Question
TPE is a commonly used buffer for DNA electrophoresis. This buffer contains 89 mM Tris, 23 mM Phosphate, 2 mM EDTA as the final concentrations. The final pH is 8.3. The 10X stock solution (10 X the final concentration) is normally prepared. You have the Tris powder (mol. wt. 121.14), Phosphoric acid H3PO4 (liquid 91.3 % pure, density 1.61 g/mL, mol. wt. 98) and a 0.5 M EDTA stock solution. You need to make up 1 L of 10X TPE. How would you make it up?
TPE is a commonly used buffer for DNA electrophoresis. This buffer contains 89 mM Tris, 23 mM Phosphate, 2 mM EDTA as the final concentrations. The final pH is 8.3. The 10X stock solution (10 X the final concentration) is normally prepared. You have the Tris powder (mol. wt. 121.14), Phosphoric acid H3PO4 (liquid 91.3 % pure, density 1.61 g/mL, mol. wt. 98) and a 0.5 M EDTA stock solution. You need to make up 1 L of 10X TPE. How would you make it up?Explanation / Answer
For a 50x mixture, we need 50x the concentrations of the 1x buffer. So we need:
50 x 40 mM Tris = 2000 mM = 2.0 M Tris
50 x 20 mM = 1000 mM = 1.0 M Acetate
50 x 1 mM = 50 mM EDTA.
Since M = mols per liter and we want to make 1.0 L of solution, we thus need 2.0 moles of Tris and 1.0 mole of acetic acid, plus the EDTA.
Since the MW of Tris is 121.14 g/mol, we need 2 * 121.14 = 242.28 g Tris powder.
Then, since the MW of acetic acid is 60 g/mol, we need 60 g acetic acid. Since the density of acetic acid is 1.05 g/mL, we need 60/1.05 = 57.14 mL acetic acid.
Finally, we calculate the amount of EDTA solution needed as:
(final volume) (final concentration)/(stock concentration)
So we need (1000 mL) (50 mM) / (500 mM) = 100 mL EDTA solution.
Thus, in order to prepare the 50x TAE buffer we need to dissolve 242.28 g Tris powder, 57.14 mL acetic acid, and 100 mL of 0.5 M EDTA stock solution in enough water to make 1 liter of solution.
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