McClean: Potassium Phosphate

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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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