LABORATORY ii

Course objectives: 

This course will enable the students to develop an understanding about normal flora from different parts of the body and analyze their metabolic capability; examine the bacterial susceptibility to various antibiotics; practical aspects of components of immune system as well as their function. The objectives of this course is also to provide students with experimental knowledge of molecular biology and genetic engineering and bioinformatics.

Course Outcomes (COs):

Experiments based on:

Immunology

1. Selection of animals, preparation of antigens, immunization and methods of blood collection, serum separation and storage.
2. Antibody titre by ELISA method.
3. Double diffusion, Immuno-electrophoresis, Radial Immuno diffusion
4. Complement fixation test
5. Isolation and purification of IgG from serum or IgY from chicken egg.
6. SDS-PAGE, Immunoblotting, Dot blot assays.
7. Blood smear identification of leucocytes by Giemsa stain.
8. Separation of leucocytes by dextran method.
9. Demonstration of Phagocytosis of latex beads and their cryopreservation.
10. Separation of mononuclear cells by Ficoll-Hypaque and their cryopreservation.
11. Demonstration of ELISPOT.
12. Demonstration of FACS.

Medical Microbiology

13. Preparation of blood smear and Enumeration of RBC and WBC

14. Isolation of Normal Flora from Skin

      15. Isolation and identification of Staphylococci and Streptococci from human body.

      16. Antibiotic sensitivity test by disc method

      17. Determination of MIC of antibiotics

      18. Normal Flora of Oral Cavity: Dental carries susceptibility test

Bioinformatics

19. Using NCBI and Uniprot web resources.
20. Introduction and use of various genome databases.
21.  Sequence information resource: Using NCBI, EMBL, Genbank, Entrez Swissprot/ TrEMBL, UniProt.
22. Similarity searches using tools like BLAST and interpretation of results.
23.  Multiple sequence alignment using ClustalW.
24.  Phylogenetic analysis of protein and nucleotide sequences.
25.  Use of gene prediction methods (GRAIL, Genscan, Glimmer).
26.  Using RNA structure prediction tools.
27.  Use of various primer designing and restriction site prediction tools.
28.  Use of different protein structure prediction databases (PDB, SCOP, CATH).
29.  Construction and study of protein structures using Deepview/PyMol.
30. Homology modelling of proteins.
31.  Use of tools for mutation and analysis of the energy minimization of protein structures.
32.  Use of miRNA prediction, designing and target prediction tools.

Molecular Biology and Genetic Engineering

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