01:694:215 Lecture Notes - Lecture 11: Uracil, Base Pair, Bacteriostatic Agent
26 Mar 2018
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Yeast: transformable, fast developmental time, well conserved, inexpensive, few introns. Auxotrophic- needs compound in media to grow. Identify mutants that fail to grow on sd-trp media. Screen for colonies that failed to grow. Null mutants- kanr (entire gene is deleted) But strong selection because only cells with the integrated fragment will grow on the antibiotic, kanamycin. Partial mutant- better to study nature of mutants. To isolate which gene is responsible for mutation: do a complementation test. Mate mutant in mata mated with mutant in mat alpha. Start with wild type trp and leu auxotroph strain. Mutate the strain and screen for cells that are defective in trp. Introduce plasmid library containing different genes into the mutant. Select for cells that picked up plasmid with leu by growing in the absence of leucine (leu, leu trp) Screen for cells that fail to grow in the absence of tryptophan. Selection- 5foa, only grow what you want (ura3 mutants)
