4-1.2.1. Growth and harvest of bacterial culture

Bacterial cell culture is more convenient than any other microbe, as it requires only liquid medium (broth) containing essential nutrients at optimal concentrations, for the growth and division of bacterial cells. The bacterial cells are usually grown on a complex medium like Luria-Bertani (LB), in which the medium composition is difficult to decipher. Later, the cells are separated by centrifugation and resuspended in 1% or less of the initial culture volume.

4-1.2.2. Preparation of cell extract

Bacterial cell is surrounded by an additional layer called cell wall, apart from plasma membrane with some species of E. coli comprising multilayered cell wall. The lysis of cell wall to release the genetic material i.e. DNA can be achieved by following ways-

•  Physical method by mechanical forces.

•  Chemical method by metal chelating agents i.e. EDTA and surfactant i.e. SDS or enzyme (e.g. lysozyme).

Lysozyme

•  Present in egg-white, salivary secretion and tears.

•  Catalyzes the breakdown of cell wall i.e. the peptidoglycan layer.

EDTA (Ethylene diamine tetra-acetic acid)

•  A chelating agent necessary for destabilizing the integrity of cell wall.

•  Inhibits the cellular enzymes that degrade DNA.

SDS (Sodium dodecyl sulphate)

•  Helps in removal of lipid molecules and denaturation of membrane proteins.
Generally, a mixture of EDTA and lysozyme is used. Cell lysis is followed by centrifugation to pellet down the cell wall fractions leaving a clear supernatant containing cell extract.

4-1.2.3. Purification of DNA

In addition to DNA, a cell extract contains significant quantities of protein and RNA which can be further purified by following methods-