McClean: Potassium Phosphate

Overview

Gomori buffers, the most commonly used phosphate buffers, consist of a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate. By varying the amount of each salt, a range of buffers can be prepared that buffer well between pH 5.8 and pH 8.0. Phosphates have a very high buffering capacity and are highly soluble in water. However, they have a number of potential disadvantages:

• Phosphates inhibit many enzymatic reactions and procedures that are the foundation of molecular cloning, including cleavage of DNA by many restriction enzymes, ligation of DNA, and bacterial transformation.
• Because phosphates precipitate in ethanol, it is not possible to precipitate DNA and RNA from buffers that contain significant quantities of phosphate ions.
• Phosphates sequester divalent cations such as Ca2+ and Mg2+

We use this buffer so several procedures in the lab, including as a buffer for storing fixed yeast cells.

Materials

• Potassium phosphate monobasic KH₂PO₄
• Potassium phosphate dibasic K₂HPO₄

Procedure

Make up the following solutions: 1M KH₂PO₄

• 68 g per 500 ml water
• warm water before adding the KH₂PO₄
• filter sterilize

1M K₂HPO₄

• 87 g per 500 ml water
• filter sterilize

To make 1M potassium phosphate, pH 7.5:

• 83.4 ml K₂HPO₄
• 16.6 ml KH₂PO₄

Other pHs (at 25°C which is approximately room temperature) can be specified using the table below:

pH	% K2HPO4 (dibasic)	% KH2PO4 (monobasic)
6	13.2	86.8
6.2	19.2	80.8
6.4	27.8	72.2
6.6	38.1	61.9
6.8	49.7	50.3
7	61.5	38.5
7.2	71.7	28.3
7.4	80.2	19.8
7.6	86.6	13.4
7.8	90.8	9.2
8	94	6

Notes

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Contact

• Megan N McClean 14:01, 02 July 2015 (EDT)

or instead, discuss this protocol.