Good morning,
does anyone know why buffered-saturated phenol is routinely used instead of Phenol:Chloroform:Isoamyl Alcohol in protocols for DNA extraction from low-melting point agarose?
Thank you in advance
Diletta Ciardo
A mixture of phenol:chloroform:isoamyl alcohol (25:24:1) is then added to promote the partitioning of lipids and cellular debris into the organic phase, leaving isolated DNA in the aqueous phase. Following centrifugation, the aqueous phase containing the purified DNA can be transferred to a clean tube for analysis.
Following articles have reported o this method
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035266/
https://geneticeducation.co.in/phenol-chloroform-dna-extraction-basics-preparation-of-chemicals-and-protocol/
Phenol is a weak acid therefore it needs to be equilibrated with a buffer ( e.g Tris) to bring the pH to a particular target—either acidic for RNA purification or slightly alkaline for DNA purification. This aids in the DNA extraction process, as this buffer saturated phenol helps denature proteins while they are still in the aqueous solution. Buffer saturated phenol has a density that is only slightly higher than that of water. So if your aqueous phase contains enough salt or any other solutes that would increase its density, then you could end up with phase inversion during extraction, where your aqueous phase is under the phenol, rather than on top of it. Chloroform is significantly denser than water, so adding it to the organic phase increases the overall density of that phase, helping to prevent phase inversion.
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