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Unformatted text preview: Lab 5: Bacterial Transformation Important: Transformation must be performed under sterile conditions. Use only autoclaved plastic-ware and always work with a flame in front of you. Also, bacteria are very labile in high calcium, so keep the bacteria on ice and away from the flame at all times to keep them viable. Objectives: Transform DNA into E. coli Learn how to transform bacteria Learn how antibiotics are used as a selective agent Learn how blue-white screening works Introduction: This is a very basic technique that is used on a daily basis in a molecular biological laboratory. The purpose of this technique is to introduce a foreign plasmid into a bacterial cell and to use those bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid, i.e. to transfer genetic information vital to the survival of the bacteria. The plasmid: A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base pairs) that contains important genetic information for the growth of bacteria. In nature, this information is often a gene that encodes a protein that will make the bacteria resistant to an antibiotic. Plasmids probably came about as a result of bacteria evolving in close proximity to other heterotrophs. Bacteria often grow in the same environment as molds and fungi and compete with them for food (complex organic material). As a result, molds and fungi have evolved to make toxins that kill bacteria (which we now use as antibiotics in medicine) in order to win in the competition for food. Bacteria, in turn, evolved to make proteins that inactivate the toxins. The bacteria share this vital information by passing it among themselves in the form of genes in plasmids. Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest. A plasmid containing resistance to an antibiotic (usually ampicillin or kanamycin) is used as a vector. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin. The bacteria are then spread over a plate that contains ampicillin. The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid. There are many different kinds of plasmids commercially available. All of them contain 1) a selectable marker (i.e., a gene that encodes for antibiotic resistance), 2) an origin of replication (which is used by the DNA making machinery in the bacteria as the starting point to make a copy of the plasmid) and 3) a multiple cloning site. The multiple cloning site (MCS) has many restriction enzyme sites (to be discussed in a later lab) and is used to insert the DNA of interest....
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This note was uploaded on 04/09/2011 for the course CHEM 4461 taught by Professor Max during the Spring '08 term at Lamar University.
- Spring '08