LABORATORY (MOLECULAR BIOLOGY AND GENETIC ENGINEERING)

Concept of lac-operon: a) Lactose induction of B-galactosidase. b) Glucose Repression. c) Diauxic growth curve of E.coli 2. UV mutagenesis to isolate amino acid auxotroph 3. Phage titre with epsilon phage/M13 4. Genetic Transfer-Conjugation, gene mapping 5. Plasmid DNA isolation and DNA quantitation 6. Restriction Enzyme digestion of plasmid DNA 7. Agarose gel electrophoresis 8. Polymerase Chain Reaction and analysis by agarose gel electrophoresis 9. Vector and Insert Ligation 10. Preparation of competent cells 11. Transformation of E.coli with standard plasmids, Calculation of transformation efficiency 12. Confirmation of the insert by Colony PCR and Restriction mapping 13. Expression of recombinant protein, concept of soluble proteins and inclusion body formation in E.coli, SDS-PAGE analysis 14. Purification of His-Tagged protein on Ni-NTA columns a) Random Primer labeling b) Southern hybridization