Presentation10 - in the middle of a reporter gene like Lac...

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Transformation of E.coli
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Introduce a foreign plasmid into a bacteria Bacteria amplifies the plasmid Natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria. What is a plasmid?
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A plasmid containing resistance to an antibiotic (usually ampicillin) is used as a vector The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate 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
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1) a selectable marker (i.e., a gene that encodes for antibiotic resistance) 1) Origin of replication 1) Multiple cloning site 1) The multiple cloning site is usually
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Unformatted text preview: in the middle of a reporter gene like Lac Z Competent Cells : DNA is a very hydrophilic molecule For bacteria to take in the plasmid, they must first be made "competent" to take up DNA This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium A cell that takes up a recombined plasmid is not be able to express the lacZ and would, in turn, not be able to produce a functional -galactosidase Conversely, a cell that has taken up non-recombined plasmid will express the lacZ- subunit and thus produce a functional -galactosidase. A cell that does not take up any plasmid is not conferred with antibiotic resistance and will die upon plating...
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Presentation10 - in the middle of a reporter gene like Lac...

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