1. Isolation Of Total Cellular DNA From
Microorganisms
Mrs.N.Gunasheela
Assistant Professor,
Department of Microbiology,
Sri Ramakrishna College of Arts & Science for Women,
Coimbatore.
2. 1. Total cell DNA : usually genomic DNA as a
source for gene cloning
2. Pure plasmid DNA
3. Phage DNA
Purification of DNA from living cells
3. Basic steps in preparation of total cell
DNA
from a culture of bacteria
4. Growing and harvesting a bacterial culture
Culture media
M9 (defined media): supplements are dependent on species
Luria-Bertani (LB) (undefined media): tryptone (amino acids), yeast extract, NaCl
Bacterial cell number
OD600 = 1 → 0.8 X 109 cells/ml
Harvesting bacteria: spin at 8,000rpm for 10 min
5. Preparation of a cell extract
•physical methods by mechanical forces
• chemical methods using chemical agents that affect the integrity of
the cell barrier
1. Lysozyme: an enzyme which digest the polymeric compounds that give the cell wall
2. EDTA: an aminopolycarboxylic acid which removes Mg2+ that are essential for
preserving the overall structure of the cell envelope and also inhibits cellular
enzymes that could degrade DNA
3. SDS (sodium dodecyl sulphate): a detergent that removes lipid molecules and
thereby cause disruption of the cell membrane
6. Purification of DNA from a cell extract
•using reagents which degrade the contaminants, leaving a pure solution of
DNA
; organic solvents – 1:1 mixture of phenol and chloroform
→ precipitates: proteins, nucleic acids in aqueous solution
enzyme – proteinase K: break polypeptides down into small units
Ribonuclease: remove the mRNA
7. Purification of DNA from a cell extract
using ion-exchange chromatography, using positively charged resin and salts
8. Concentrations of DNA samples
Ethanol precipitation in the presence of salt (Na+) and at a -20C
9. Measurement of DNA concentration
•can be accurately measured by ultraviolet absorbance
spectrophotometer
• A260 absorbance of 1 = 50 ug/ml
• Good DNA purity: A260/A280 > 1.8
10. Other methods for the preparation of total cell DNA
The CTAB method for purification of plant DNA
Grinding frozen
plant cells
DNA purification by the guanidinum thiocyanate
and silica method
