Isolation and Purification of DNA (46 mins) | Lecture 46/90 | ASRB NET (ICAR-NET) Veterinary Microbiology

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Techniques in Microbiology and VaccinologyPrinciple of Molecular Techniques

The procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages:

A culture of bacteria is grown and then harvested.

The cells are broken open to release their contents.

This cell extract is treated to remove all components except the DNA.

The resulting DNA solution is concentrated

Most bacteria can be grown without too much difficulty in a liquid medium (broth culture).

M-9 is an example of a defined medium in which all the components are known.

Luria-Bertani (LB) is a complex or undefined medium, meaning that the precise identity and quantity of its components are not known.

For preparing cell extract the chemicals that are used depend on the species of bacterium involved, but with E. coli and related organisms, weakening of the cell wall is usually brought about by lysozyme, ethylenediamine tetraacetate (EDTA), or a combination of both.

Usually, a detergent such as sodium dodecyl sulphate (SDS) is also added.

For purification organic solvents like phenol + chloroform is added or ion exchange chromatography is done.

The DNA is then concentrated using ethanol.