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Lecture 14

BIOL 5060 Lecture 14: 3:28_RecDNATech_Hoffman

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Boston College
BIOL 5060

3/28_RecombinantDNATechnology 3/28/2017 7:19:00 PM What protein binds to this sequence? • We have knowledge of the cis element not the trans • One hybrid (budding yeast) o There are 2 reporters o This sequence has a His3 reporter ▪ We want the new plasmid to be able to have GAD protein bind ▪ Not really interested in proteins function, just if it can bind ▪ Transform yeast to leu+ media library ▪ Screen for reporter expression ▪ Xgal blue colinies ▪ Insert cDNA in leu+ media to make a library o Very little His3 expression can produce a His+ colony (so we have to make sure there aren’t false positives?) ▪ 3- aminotriazole His3 inhibitor ▪  addition of 3AT to this medium requires higher level of expression for growth to occur When is my protein bound to the DNA in cells? • Want to make sure we analyze in vivo • Chromatin Immunoprecipitation (ChIP) o Take cells and treat with formaldehyde to create covalent bonds (cross link) between proteins and the DNA to which they are bound o Isoalte DNA o Physically shear to ~500 bp o Immune precipitate protein of interest (need Ab) o Reverse crosslink (to free DNA from protein) o Detect region of interest vs. control region of DNA look for enrichment of region of interest • 1) shear DNA IP  there are some random sequences that make is through a control is oligos around gene o  those that don’t make it through go through PCR o Controls: oligos to a distant site, sheared chromatin without IP • 2) Endpoint PCR- a specific number of
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