LAB1.NEW - Recombinant DNA Session 1 Transformation of E...

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Recombinant DNA Session 1: Transformation of E. coli BACKGROUND Getting foreign DNA to propagate in E. coli is essential to cloning genes. Because these extrachromosomal elements (episomes) are smaller than the bacterial chromosome, they are easy to purify away from the mass of chromosomal DNA Some elements, especially the plasmids, can be made to replicate in hundreds of copies more than the bacterial chromosome itself, providing a large relative amplification of the copy number of the cloned gene. In this session, we'll see how to put a small circular plasmid molecule into an E. coli strain that has no plasmid The plasmid has two important properties for today's experiment 1. It replicates independently of the bacterial chromosome 2. It carries a gene for drug resistance that is not found on the bacterial chromosome. One of the plasmid molecules that you will use has also been previously engineered to contain foreign genetic material (an insert). Bacteria that take up the plasmid will become drug resistant while the bacteria that haven't been able to take up the plasmid DNA will be killed by an antibiotic has been added to the Petri plates that you will use. Only the bacteria containing the plasmids with drug-resistance genes will survive. E. coli is a gram-negative bacterium, much studied because it is a common gut bacterium of man and because genetic crosses of chromosomal DNA between different strains can be done to map genes along the chromosome. Unfortunately, E. coli does not readily take up DNA without special treatment. During much of the early history of recombinant DNA, treatment with divalent cations such as calcium and heat shock was used to force E. coli to take up recombinant DNA. Now, high voltage shock (electroporation) can be used to shoot DNA through the membrane of E. coli cells. Electroporation is the most efficient method of all for E. coli cells but requires a machine costing several thousand dollars. Because the divalent cation method is the most generally useful and simple method, you'll learn that method. Parameters of the divalent cation method of transforming cells. The exact way that it works is unknown. The following considerations are important. Bacterial cells that are still in the middle of their exponential growth phase must be used. There is something different about the structure of the bacterial cell wall and/or cell surface membrane in stationary cells arrested in growth that prevents DNA uptake. Many protocols use chilled cells and preincubate with divalent cations such as calcium. The chilling is thought to "freeze" the cell membrane lipids. The divalent cations bind to the
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