McClean: Potassium Phosphate: Difference between revisions
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==Overview== | ==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== | ==Materials== | ||
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==Procedure== | ==Procedure== | ||
Make up the following solutions: | |||
''1M KH<sub>2</sub>PO<sub>4</sub>'' | |||
68 g per 500 ml water | |||
warm water before adding the KH<sub>2</sub>PO<sub>4</sub> | |||
filter sterilize | |||
''1M K<sub>2</sub>HPO<sub>4</sub>'' | |||
87 g per 500 ml water | |||
filter sterilize | |||
To make 1M potassium phosphate, pH 7.5: | |||
83.4 ml K<sub>2</sub>HPO<sub>4</sub> | |||
16.6 ml KH<sub>2</sub>PO<sub>4</sub> | |||
==Notes== | ==Notes== | ||
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==Contact== | ==Contact== | ||
*'''[[User:Megan N McClean|Megan N McClean]] 14:01, | *'''[[User:Megan N McClean|Megan N McClean]] 14:01, 02 July 2015 (EDT)''' | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
Revision as of 07:59, 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
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.
