Lecture 20: Gel Electrophoresis
Be familiar with the 3 forms of plasmid DNA and how they migrate on agarose gels. Understand the relationship between migration rate and concentration of agarose/acrylamide and molecular mass. Be able to interpret the results of a protein acrylamide gel with regard to subunit composition of a polypeptide (e.g. the effects of reducing agents, or SDS). Be able to explain how DNA or proteins can be visualized. Understand the use of gel electrophoresis (i.e. what is it useful for?)
Lecture 21: DNA Sequence Analysis
Be able to explain the method of dideoxy sequencing. Be able to provide the original template sequence if supplied the results of a sequencing gel.
Lecture 22: Polymerase Chain Reaction
Be able to explain how the difference PCR products are amplified, i.e. original template, products with indeterminant 3' ends, and products with determinant 5' and 3' ends. Be able to explain what occurs during a single PCR cycle, and what the essential components are.
Lecture 23: PCR, cont.
Be able to determine the different estimated primer melting temperatures given an example primer. Understand the effects of magnesium and temperature on primer/template annealing. Be able to determine the extinction coefficient for a supplied primer. Be able to identify features of designed primers which may lead to problems during a PCR experiment (i.e. a sequence which might produce secondary structure in the primer, etc.)
Lecture 24: Cloning PCR Products
Be able to diagram and explain how you would use the PCR method to create a point mutation, insertion, deletion or gene fusion. Understand what a "half-site" is and how to use it in PCR cloning. Understand what a "mega-primer" is, what asymmetric PCR is, what inverse PCR is.
Lecture 25: Prokaryotic Expression Vectors
Understand how alpha complementation works, and how it can be used to identify plasmids with inserts. Understand how expression in the pET and pTrcHis vectors is regulated. Understand how a His tag works.
Lecture 26: Protein Sequencing, Peptide Mapping, Synthetic Genes
Understand how N- and C- terminal sequencing is done, and the limitations of both. Understand how peptide mapping is performed, and if you are provided with data from such an experiment propose an alignment of peptide fragments. Understand how you could assign disulfide bond assignments. Understand the principle of how to construct a synthetic gene.
Lecture 27: cDNA Libraries
Understand how to make a cDNA library. Be able to explain the use of C-tailing and linkers in the construction of such a library.
Lecture 28: Genomic Libraries
Be able to determine how large a library you will need for a given probability of finding a gene of interest. Understand how phage lambda is used to construct genomic libraries. Be able to explain the terms antigen, antibody and epitope.
1998 Dr. Michael Blaber