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Lecture 11 • Assignment 4 • Notebook • Beta-galactosidase assay • What do you want me to talk about in the review? Experiment 3 • Remember you have to prepare in your notebook the volumes you are going to use for your restriction digestion. Assignment 4 • PART 1: Experiment 3 • Page 1 Will present 3 labeled gels: 1 the gel of the digest of the vector and digest for DNA fragment isolation; 2 the gel of the ligation reactions and isolated DNA fragment; 3 the gel of the restriction digestions of the two clones. • Page 2 You will summarize the success or failure of each step of the process of cloning. • PART 2: Experiment 5 • Page 3-4 will be your report on the yeast mutant screen. The text following the table shall answer four questions about the mutants you characterized. Summary of Expt. 5 in your notebook 1 2 3 4 5 6 7 8 P Initial Phenotype of Mutant Complementation Test beta-galactosidase Test Probable Mutation Red or White Red or White Blue or White swi5, swi, she or unknown Notebook • Look at the instructions and expectations on the web site. • Goal is to have a document that you can show a potential employer or supervisor demonstrating what you have experience doing and that you have the ability to record data in a true functioning lab notebook. The LacZ Reporter Gene beta-galactosidase is an enzyme encoded by the bacterial gene lacZ. beta-galactosidase cleaves the colorless substrate X-gal (5bromo-4-chloro-3-indolyl-galactopyranoside) into galactose and a blue insoluble product of the cleavage. Therefore, lacZ can be used as a reporter gene (blue/white selection) X-GAL Cis-acting elements URS 1 URS 2 lacZ GAL1-10 URS 2 lacZ Cis-acting elements lacZ Mother daughter URS 2 lacZ Late G1 in both mothers and daughters GAL1-10 lacZ URS 1 Galactose inducible in both mothers and daughters. Apart the elements are independent; together the elements are restrictive and not additive. Properties URS 1 URS 2 lacZ *Haploid specific *Cell cycle regulated Mother cell specific GAL1-10 URS 2 *Haploid specific *Cell cycle regulated Galactose inducible lacZ Trans-acting factors required for expression Cis element URS1 Trans acting factors Swi5p Swi1p, Swi2p, Swi3p URS2 Swi4p, Swi6p Swi1p, Swi2p, Swi3p GAL1-10 Gal4p Therefore in swi5lf or shelf mutant haploid cells grown on Galactose plates: URS 1 URS 2 CAN1 Is not expressed GAL1-10 URS 2 lacZ Is expressed BLUE Therefore, in the other swi mutant haploids grown on Galactose plates URS 1 URS 2 CAN1 Is not expressed GAL1-10 URS 2 lacZ Is not expressed WHITE Summary of Expt. 5 in your notebook 1 2 3 4 5 6 7 8 P Initial Phenotype of Mutant Complementation Test beta-galactosidase Test Probable Mutation Red or White Red or White Blue or White swi5, swi, she or unknown What would you like me to explain to you now, and what would you like me to review next lecture?