BMB 442Laboratory in Proteins, Nucleic Acids, and Molecular Cloning

3 credits · Biochemistry and Molecular Biology · Penn State

Laboratory in enzyme purifications and assay techniques; nucleic acid isolation and characterization, including plasmid preparation. BMB 442 / MICRB 442 Laboratory in Proteins, Nucleic Acids, and Molecular Cloning (3)The DNA portion of B M B/MICRB 442 serves as an introduction to fundamental techniques of recombinant DNA technology and as a reinforcement of principles of Molecular Genetics from lecture courses. The central experiment entails all basic procedures necessary to clone a gene, i.e. to make a recombinant molecule comprised of DNA from two sources. Students use restriction enzymes to cut two distinct DNA molecules into smaller fragments. The fragments are mixed and treated with the enzyme Ligase, which randomly combines small fragments into large recombinant DNA molecules in new combinations different in composition from either original molecule. The recombinant molecules, which include genes that confer drug resistance, are transformed intoE. coli cells that initially have no drug resistance. Cells that acquire recombinant DNA molecules are identified by selective plating on growth media containing drugs. From the transformed cells, recombinant DNA is isolated and analyzed by agarose gel electrophoresis, completing the array of basic gene cloning techniques. In addition to this central, multi-session experiment, students also do PCR and an investigation of the lac operon, a classic molecular genetic model system. The proteins portion of BMB 442 / MICRB 442 is designed to introduce students to protein biochemistry topics and laboratory techniques typically encountered in academic and commercial settings. Students will learn about buffers, spectroscopy, enzyme purification and characterization methods. Specifically, the experiments include preparation of buffers and performing kinetic studies to determine Km and Vmax values. Separation of a mixture of phycobiliproteins using ion-exchange column chromatography is a major experiment that the students will perform to learn protein purification methods. In this experiment they will learn how to pour a column, apply sample, elute it with salt gradient and collect fractions using automated fraction collector. Ammonium sulfate precipitation and dialysis will be part of protein purification procedures. Characterization of the separated proteins will be performed by determining the absorption spectra with spectrophotometer and by determining the molecular weights of the subunits of the phycobiliproteins by SDS-polyacrylamide gel electrophoresis.