1. BTE 204: Fundamentals of Genetic Engineering
Sadia Sayed

2. How to manipulate the gene?
Target a gene: Gene of Interest (GOI)
Purify DNA from living cell
Flow chart of Genetic Engineering
Manipulation of purified DNA
Introduction of DNA into living cell
Harvesting product of interest

3. phage DNA will be needed if a phage cloning vector is to be used.
The genetic engineer will, at different times, need to prepare at least three distinct kindsof DNA.
total cell DNA (consists of the genomic DNA of the organism along with any additional DNA molecules,such as plasmids, that are present).
plasmid DNA
phage DNA will be needed if a phage cloning vector is to be used.

4. Purification of DNA from Living cells4

5. Growing and harvesting a bacterial culture5

6. 6

7. Growing and harvesting a bacterial culture
Lysozyme
EDTA: removes magnesium ions that is required for integrity of the cell membrane and also chelates MgCl2 and thereby inhibits Dnase.
SDS/CTAB/Lauryl Sarcosinate: remember detergents wash lipid substances!
NaOH: causes hydrolysis of the membrane (alkaline lysis)
NaCl: causes the cell to rupture 7

8. Purification of DNA from cell extract (Get rid of protein!)
1:1 Phenol Chloroform mixture: The organic layers captures the protein. The aquous layer has the DNA.
DNA is charged negatively, so forms hydrogen bond with water.
Also protease can be added to degrade protein contanmination. 8

9. Precipitate DNA from aqueous layer
Alcohol addition: Isopropanol or ethanol addition causes the polarity of the solution to change. So DNA precipitates! 9

10. CTAB method of DNA isolation

11. DNA purification by Guanidium thiocyanate method

12. Measurement of DNA concentration
260/280 nm Absorbance: 1.8 for pure DNA;
A260 = 50µg of dsDNA/ml 12

13. Purification of Plasmid DNA
Separation on the basis of Size: Gel electrophoresis; supercoiled will migrate towards negative voltage much faster than the open circular . 13

14. Purification of Plasmid DNA: Steps
Lysis of the cell: Lysis solution 1 14

15. Purification of Plasmid DNA: Steps
Lysis of the genomic DNA: Lysis solution 2 and 3; Alkaline denaturation
Apply NaOH and then glacial acetic acid 15

16. Purification of Plasmid DNA: Steps
Density gradient centrifugation: Alternative to Phenol-chloroform wash
Buoyant density of DNA is 1.7g/cm3 16

18. Purification of Plasmid DNA: Steps18

19. Plasmid amplification

20. How do we view DNA?
Agents that bind to DNA and also illuminates after UV exposure: EtBr (Intercalating agent)
But the DNA after EtBr exposure is not usable without column purification 20

21. Prepation of bacteriophage DNA

22. Preparation of λ phage DNA
Inducers of Lysogeny and lytic: cI protein= lysogeny
Balance between bacteria and phage is important 22

23. Preparation of Bacteriophage DNA
Inducers of Lysogeny and lytic: cI protein= lysogeny
Balance between bacteria and phage is important 23

24. Preparation of Bacteriophage DNA
Collection of phage particle in PEG 24

25. Preparation of M13 DNA 25