1. Purification of DNA from living cells
Total cell DNA : usually genomic DNA as a source for gene cloning
Pure plasmid DNA
Phage DNA

2. The basic steps in preparation of total cell DNA
from a culture of bacteria

3. 3.1.1 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

4. 3.1.2 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
EDTA (ethylenediamine tetraacetate)

5. 3.1.3 Purification of DNA from a cell extract
- by 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

6. 3.1.3 Purification of DNA from a cell extract
by using ion-exchange chromatography
; using positively charged resin and salts

7. 3.1.4 Concentrations of DNA samples
Ethanol precipitation in the presence of salt (Na+) and at a -20C

8. 3.1.4 Measurement of DNA concentration
- can be accurately measured by ultraviolet absorbance spectrophotometry
- A260 absorbance of 1 = 50 ug/ml
- Good DNA purity: A260/A280 > 1.8

9. 3.1.4 Other methods for the preparation of total cell DNA
Grinding frozen
plant cells
The CTAB method for purification of plant DNA
DNA purification by the guanidinum thiocyanate
and silica method

10. 3.2 Preparation of plasmid DNA
3.2.1 Separation on the basis of size

11. 3.2.2 Separation on the basis of conformation

12. Plasmid purification by the alkaline denaturation method

13. Caesium chloride (CsCl) density gradient centrifugation

14. Linear & open circular DNA: decrease in a buoyant density by 0
Linear & open circular DNA: decrease in a buoyant density by 0.125g/cm3
Supercoiled DNA: decrease in a buoyant density by 0.125g/cm3

15. Purification of plasmid DNA by Et-Br-CsCl density gradient centrifugation

16. Plasmid amplification
Chloramphenicol: an inhibitor of protein synthesis
→ plasmids continue to replicate / chromosome and cell division are blocked