Lecture 2-3 - Lecture 3 Mutagenesis The Scientific Method...

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Lecture 3 Mutagenesis & The Scientific Method 2/21/11 © Nicola Davies 2011 Site Directed vs. Random Mutagenesis & The Scientific Method Lecture 3 February 21, 2011 2 Site Directed Mutagenesis: 1 Method PCR 2 PCR 1 PCR 3 3 Site Directed Mutagenesis: 1 Method yfg lacZ Amp R Cloning Vector lacZ Ampicillin Resistance MCS PCR 2 PCR 1 PCR 3 + = 4 Site Directed Mutagenesis: 1 Method yfg lacZ Amp R yfg lacZ Amp R Cam R + = Restriction Endonuclease Digest Chloramphenicol Resistance Restriction Endonuclease Digest + Amp R sacB ori * = Amp R sacB ori * yfg Cam R 5 Site Directed Mutagenesis: 1 Method Amp R sacB ori * Homologous Recombination yfg Cam R Bacterial chromosome 6 Enrich for second recombinations by growing in sucrose Two possible sites of homologous recombination Recombine at site A: regenerate WT Recombine at site B: generate a mutant strain 10% sucrose is lethal to strains carrying the sacB gene Patch to find colonies that are Cam R , Amp S A A B B Site Directed Mutagenesis: 1 Method Bacterial chromosome containing mutated copy of yfg
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