Molecular Immunology Module
Lab 1
Lecture: Purification of genomic DNA
Laboratory: Extraction and purification of B cell genomic DNA
Lab 2
Lecture: Overview of DNA module and Introduction to V(D)J recombination.
Laboratory: Set up restriction digests, make agarose gels and test restriction digestion.
Lab 3
Lecture: Southern blotting.
Laboratory: Gel electrophoresis (started Tuesday p.m.) and Southern transfer done in class.
Lab 4
Lecture: Genomic phage libraries and DNA cloning vectors.
Laboratory: Pre-hybridize and hybridize filters from Southern transfer. Set up digestions of plasmid DNA for restriction mapping.
Lab 5
Lecture: Restriction mapping.
Laboratory: Wash filters (done by staff) and detect hybridized bands on Southern blots. Run gels to analyze digests of plasmid DNA for mapping. Set up digestions to isolate insert and vector for subcloning.
Lab 6
Lecture: Subcloning.
Laboratory: Separate digests in agarose gel. Purify DNA and set up ligations
Lab 7
Problem Set on Analysis of Ig Gene Rearrangement by Southern Hybridization due.
Lecture: Plasmid miniprep.
Laboratory: Transform bacteria.
Lab 8
Lecture: DNA sequencing: Maxam & Gilbert and Sanger's methods.
Laboratory: Mini-prepping of plasmid DNA and analysis by restriction digestion. Preparation for sequencing reactions.
Lab 9
Problem Set on restriction mapping and subcloning due.
Lecture: Antibody DNA sequence data and bioinformatics.
Laboratory: Set up the first PCR reaction. Read sequencing data and work on problem set.
Lab 10
Problem Set on DNA sequencing due.
Lecture: PCR as a general technique and in analysis of V(D)J recombination.
Laboratory: Set up the second PCR reaction. Run agarose gel.
Lab 11
Lecture: Summary of DNA module.
Review and questions
Laboratory: Analyze PCR data and complete problem set in lab.
