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Experiment 1d
ASSAY FOR INSERT TRANSFORMANTS
Today, you will determine if your bacteria have become transformed by your
recombinant plasmid. To accomplish this, you will perform the following two assays. The first
is very simple: look on your plates for growing colonies. If there are colonies, congratulations!
You have successfully transformed bacteria. This, of course, is true because the presence of a
plasmid within the bacteria has allowed growth under the selective pressure of an otherwise
lethal dose of antibiotic (e.g. ampicillin).
The second assay is designed to detect the presence of bacteria that have the gene (in the
plasmid we have made) for and therefore can express the HPPD protein. This involves
observing the development the unique color associated with the oxidative breakdown of the
product of the HPPD reaction, homogentisate (see lab manual introduction).
