19 BIO 326R screening - BIO 326R Mutations and screening...

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BIO 326R Mutations and screening
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Selection – auxotrophs and prototrophs Finding a Phe- mutant from a “wild type” or prototroph population Must find a mutant that grows on minimal media with phenylalanine supplementation, but does not grow on regular minimal media (no phenylalanine) This is a phe- phenotype by definition Mutagens + massive screening (replica plating) Mutagens aren’t strictly necessary, but can reduce the number of colonies that must be screened by a large amount Will likely need to grow thousands of colonies in any case… (genomes are big). There may be many mutations, but only a few will cause the phe- phnotype that you are seeking.
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Finding a Phe -  mutant How many colonies must be screened?   (did you add a  mutagen?)   Minimal media +Phenylalanine Minimal media (no Phenylalanine added) Mutant Fails to grow in absence of Phenylalanine Grow on MM + phe, then replica plate colonies over to MM
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Finding reversion mutations Selecting a Phe+ revertant from a Phe- population Starting with Phe - (auxotrophic) culture, plate on media without Phenylalanine, any growth is due to reversion mutants Similar strategy for resistance (Amp+) mutants i.e. take strain known to be sensitive, grow large amounts, plate on media with Ampicillin – look for growth
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Finding a Phe -    Phe +  revertant (easy!) Minimal media (no phenylalanine) Starting with Phe - auxotroph strain (perhaps made as in previous slide) Grow up large culture (include Phenylalanine) add mutagen/irradiate. Plate ~10 7 or more cfu on minimal media (no phenylalanine), ONLY Phe+ should grow
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Replica plating The initial plating must be done on the supplemented media (media that allows the mutant to grow) Colonies are then replica plated to minimal media that selects against the mutant Remember that you need to allow the mutant strain/colony to grow before you can replica plate it. Therefore: Plating both at the same time won’t work. In order for replica
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This note was uploaded on 04/09/2009 for the course BIO 326R taught by Professor Whiteley during the Spring '08 term at University of Texas at Austin.

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19 BIO 326R screening - BIO 326R Mutations and screening...

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