Isolation & Purification of DNA

Isolation and Purification of Chromosomal DNA,Plasmid DNA,Bacteriophage DNA used in Recombinant DNA Technology or Biotechnology to produce Recombinant DNA or Desired DNA

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

3. B asic steps in preparation of total cell DNA f rom 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 A 260 absorbance of 1 = 50 ug /ml Good DNA purity: A 260 /A 280 > 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 a nd silica method

11. Preparation of plasmid DNA Separation on the basis of size

12. Separation on the basis of conformation

13. Plasmid purification by the alkaline denaturation method

14. Caesium chloride ( CsCl ) density gradient centrifugation

15. Linear & open circular DNA: decrease in a buoyant density by 0.125g/cm 3 Supercoiled DNA: decrease in a buoyant density by 0.125g/cm 3

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

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

18. Isolation and Purification of bacteriophage DNA

19. Purification of M13 phage DNA