304. Molecular Cloning (EXP-304)

This two part exercise provides state-of-the-art information and practical experience with a variety of techniques that form the foundation of the biotechnology industry. In part A, students create a strain of E. coli that is resistant to the antibiotic ampicillin by introducing a plasmid that contains an ampicillin-resistance gene. The success of the transformation is monitored by growing the bacteria on an ampicillin-containing media. This experiment provides sufficient sterile materials for sixteen platings. In Part B, students isolate the amplified plasmid from the bacteria without the use of toxic chemicals. Then they digest the isolated DNA with EcoR1 and compare the electrophoretic properties of linear and circular plasmid DNA molecules. By inserting desired DNA segments into plasmids, this procedure has enabled scientists to amplify more than 1000 specific genes including those for human interferon, insulin, and growth hormone. Part B of the exercise requires a centrifuge that can be operated at a force of at least 3,000 x g (such as a microcentrifuge or a larger floor model). The entire exercise requires three 2-3 hour laboratory periods and the materials provided are shown.