PPTH 103 LAB - Isolation of Bacterial Genomic DNA

DNA extraction

process of isolation and purification of DNA from sample using combination

of physical and chemical methods.

-amplification of genes

-determine DNA

-elucidate genetic diversity

Isolation and purification

of DNA are a crucial step

in several DNA

molecular techniques

Cell Lysis
Separation of DNA from other cell components

Precipitation of DNA from other cell components

Three Basic Steps

Temperature treatment
Use of detergents
Use of enzymes

Cell membrane is disrupted

by any of the following

methods:

proteinase K.

nucleases are

inactivated by enzymes such as

Temperature treatment

use

of heat to increase fludity or

improve solubility

Use of detergents

lipids are broken down with

surfactants

Use of enzymes

proteins

incuding nucleases are

inactivated by enzymes such as

proteinase K.

Treatment with concentrated

salt solution-

Cetyl trimethylammonium

bromide (CTAB)

phenol:chloroform:isoamyl

After centrifugation

Separation of DNA from

other cell components

Treatment with concentrated

salt solution-

makes the cellular

debris like proteins, lipids,

etc. to clump together

Cetyl trimethylammonium

bromide (CTAB)

precipitates

CW debris, polysaccharides

and denatured proteins

phenol:chloroform:isoamyl

separates proteins and

polysaccharides from nucleic

acids in the cell.

organic phase

: bottom layer

aqueous phase:

upper layer

containing the DNA

Alcohol precipitation-
other cell components

• Dissolution/Resuspension of

DNA

Precipitation of DNA from

other cell components

1. Commercially available DNA

   extraction kits

2. Boiling method

Other methods of DNA Extraction