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Preparation of Equilibrated Phenol




 Through this experiment you will able to understand the preparation of equilibrated phenol and its role in the biological or molecular experiments.




Phenol, also known as carbolic acid, is an organic compound with the chemical formula C6H5OH. It is a white, crystalline solid.  It has to be handled with care because it causes white blistering to the skin immediately. The crystals are often rather wet and discolored.  Phenol contains a phenyl group which is  bonded to a hydroxyl (-OH) group. Phenol used in DNA/RNA extraction to removes non-polar proteins, lipid residues etc.


Most batches of commercial liquefied phenol are clear and colourless and can be used in molecular cloning without re-distillation. Occasionally, batches of liquefied phenol are pink or yellow, and these should be rejected and returned to the manufacturer. Crystalline phenol is not recommended because it must be redistilled at 160°C to remove oxidation products, such as quinones, that cause the breakdown of phosphodiester bonds or cause cross-linking of RNA and DNA. Before use, phenol must be equilibrated to a pH of >7.8 because the DNA partitions into the organic phase at acid pH.



Phenol readily undergoes oxidation and must therefore be redistilled before use. Since redistilled phenol is acidic, a neutralization step with Tris base and then an equilibration step with TE (pH 8.0) or a similar buffer must be performed. Equilibrated phenol is stored at -20 °C.




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