McClean: Potassium Phosphate: Difference between revisions
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Make up the following solutions: | Make up the following solutions: | ||
''1M KH<sub>2</sub>PO<sub>4</sub>'' | ''1M KH<sub>2</sub>PO<sub>4</sub>'' | ||
68 g per 500 ml water | *68 g per 500 ml water | ||
warm water before adding the KH<sub>2</sub>PO<sub>4</sub> | *warm water before adding the KH<sub>2</sub>PO<sub>4</sub> | ||
filter sterilize | *filter sterilize | ||
''1M K<sub>2</sub>HPO<sub>4</sub>'' | ''1M K<sub>2</sub>HPO<sub>4</sub>'' | ||
87 g per 500 ml water | *87 g per 500 ml water | ||
filter sterilize | *filter sterilize | ||
To make 1M potassium phosphate, pH 7.5: | ''To make 1M potassium phosphate, pH 7.5'': | ||
83.4 ml K<sub>2</sub>HPO<sub>4</sub> | *83.4 ml K<sub>2</sub>HPO<sub>4</sub> | ||
16.6 ml KH<sub>2</sub>PO<sub>4</sub> | *16.6 ml KH<sub>2</sub>PO<sub>4</sub> | ||
Other pHs (at 25°C which is approximately room temperature) can be specified using the table below: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''pH''' | |||
| align="center" style="background:#f0f0f0;"|'''% K2HPO4''' | |||
| align="center" style="background:#f0f0f0;"|'''% KH2PO4''' | |||
|- | |||
| ||(dibasic)||(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 | |||
|- | |||
| | |||
|} | |||
Revision as of 08:07, 2 July 2015
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 | % KH2PO4 |
| (dibasic) | (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
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Contact
- Megan N McClean 14:01, 02 July 2015 (EDT)
or instead, discuss this protocol.
