Isolating and Purifying Genomic DNA of Rhizobia Using a Rapid Small-Scale Method

Abstract

When Gram-negative bacteria are exposed to lysozyme, the integrity of the outer cell wall is affected by hydrolytic cleavage of complex polysaccharides. Some of the cell wall remains and the cell becomes spherical, protected mainly by the cell membrane. These cells are called spheroplasts and can be lysed to release the DNA by adding ionic detergents such as sodium dodecyl sulfate (SDS) or sodium N-lauroylsarcosine (sarkosyl). This is followed by phenol extraction to remove the proteins from the nucleic acids. Phenol denatures proteins readily, but does not completely inhibit ribonuclease activity. Guanidine isothiocyanate disrupts the cell membrane and causes rapid protein denaturation. Also, the guanidinium cation and the isothiocyanate anion inactivate the ribonucleases by disrupting their tertiary structure.