Lab Techniques for Midterm 2

Lab Techniques for Midterm 2 - Technique Yeast TwoHybrid...

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Technique How does it work? Important Reagents When would you use this? What can it tell you? Yeast Two- Hybrid (Y2H) Harnessing the independent domain structure of TFs to genetically screen for protein-protein/protein- DNA interactions. Two protein expression plasmids, reporter plasmid (‘Directed’ Y2H- when you attach bait and prey to proteins you already know.) Testing the regulation of Pol II transcription. Testing activation of downstream reporter genes. (A cDNA library can be used in place of the prey to test interactions with known proteins.) Whether two proteins interact. Whether a protein interacts with a given DNA sequence. Keywords: bait, prey, reporter, activation, DBD, AD ChIP 1) in living cells, crosslink DNA and proteins with formaldehyde . 2) Break open cells and fragment DNA into small (~100-500bp) pieces. DNA in the form of chromatin, formaldehyde, nuclease or sonicator, primers for PCR To determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. Whether a specific protein is associated with a particular DNA sequence in vivo. Limited resolution. 3) Immunoprecipitate protein (co-IP
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This document was uploaded on 03/14/2011.

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Lab Techniques for Midterm 2 - Technique Yeast TwoHybrid...

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