ISOLATION OF

CHROMOSOMAL DNA
Sources:
Microorganisms
Plants
Animals
4 stages in isolation process of
bacterial chromosomal DNA :

• Cultivation and harvesting of

bacterial cell
• Cell lysis
• Purification of chromosomal DNA
from cell extract
• Concentration of DNA
 1. The growth and harvesting of bacterial cell

 Cell is grown on appropriate medium.

 The cell growth is measured and monitored by OD

(optical density) measurement

 Cell is harvested on certain exponential phase by

centrifugation
2. The preparation of cell extract
- Cell lysis using lisozime, EDTA and SDS
- Removal of supernatan from cell debris
3. Purification of chromosomal DNA from cell extract

The result of stage 2 will contain DNA, RNA and

protein. Therefore, DNA need to be purified from
other constituents.

Purification can be done with:

 Ion exchange
 Extraction with organic solvents and enzimatically
4. DNA concentration:
The DNA concentration DNA can be done by
adding cold absolute ethanol
The determination of DNA concentration

 Done by UV-VIS Spectrophotometer UV at  260

nm, where absorbance 1,0 is equal to 50 μg double
stranded DNA/ml.

 The purity of DNA can be determined by its

A260/A280 value. If the ratio is smaller than 1.8, the
DNA is not pure enough (still contain protein or
phenol as contaminants).