Phenol–chloroform extraction is a liquid-liquid extraction technique in biochemistry and molecular biology for purifying nucleic acids and eliminating proteins. In brief, aqueous samples are mixed with equal volumes of a phenol:chloroform mixture. After mixing, the mixture is centrifuged and two distinct phases are formed, because the phenol:chloroform mixture is immiscible with water. The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The proteins will partition into the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. The upper aqueous phase is pipetted off and care is taken to avoid pipetting any of the organic phase or material at the interface. This procedure is often performed multiple times to increase the purity of the DNA.
If the mixture is acidic, DNA will precipitate into the organic phase while RNA remains in the aqueous phase due to DNA being more readily neutralised than RNA. PRINCIPLE OF EXTRACTION'' Genomic DNA is the total DNA of the plant,microorganism or any living cell.The genome is present in the nucleus where it is safe guarded by nuclear membrane from exposure to the cytoplasm.Hence it is necessary to break the cell for releasing the DNA which may be done by physical or by biochemical means,by the solubilization of membrane using detergents or by enzymes since plant cells having both cell wall and cell membrane.They must be removed one by one to release the cell contents.This can be achieved by combaining two techniques.A technique is lyse the cell gently and other is to solubilize the DNA by enzymatic or chemical methoda to remove contaminated proteins inorder to extract the DNA.
- Zumbo, P. "Phenol-chloroform Extraction" (PDF). Weill Cornell Medical College. Retrieved 19 September 2014.
|This molecular biology article is a stub. You can help Wikipedia by expanding it.|