Microbial DNA Extraction, Purification, and Quantitation

Abstract

Cell wall of microorganisms is broken by chemical or enzymatic lysis or a combination of both. Generally lysozyme is used to digest the rigid cell wall structure which has high amounts of lipid while detergent like SDS solubilizes the phospholipids in the cell membrane. EDTA destabilizes the cell envelope and deactivates the DNases by chelating with the magnesium ions in the membranes which are essential for integrity of cell envelope. Insoluble cell debris is removed via centrifugation, leaving an upper aqueous suspension containing the DNA, proteins, and RNA. Purification of DNA from proteins can be achieved by various methods, generally by protease treatment, to hydrolyze the proteins resulting in water-soluble amino acids or shaking the aqueous suspension with phenol chloroform. The aqueous phenol emulsion is then separated by centrifugation. Proteins (having both hydrophobic as well as hydrophilic amino acid residues) get collected at the interphase. While RNA can be removed by RNase treatment, DNA can be concentrated by addition of ice-chilled ethanol or isopropanol and precipitated DNA is collected as pellet by centrifugation. This chapter describes the protocol to check the purity and quantify DNA.