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Quiz#7 LC710 10/18/10 name___________ Q1 :I have a protein extract made from 1000 different mRNAs (I also have cDNAs for them). I know this extract contains a single DNA binding protein. I decide to first figure out what sequence that protein binds and then use this information to Identify the protein. First, I set up a SELEX screening system with the following paradigm. I have two outer sequences of 18 nucleotides, followed by two palindromic sequences and then a degenerate core set of 6 nucleotides (1 strand is depicted) . 5’-CATTCAAATATGTATCCGAGCTAGCTNNNNNNAGCTAGCTGATAAATGCTTCAATAAT-3’ After multiple rounds of selection, I obtain the following sequencing results 5’-CATTCAAATATGTATCCGAGCTAGCTTAATTGAGCTAGCTGATAAATGCTTCAATAAT-3’ 5’-ATTATTGAAGCATTTATCAGCTAGCTCAATTAAGCTAGCTCGGATACATATTTGAATG-3’ (1pt) What is the 6 nucleotide consensus sequence: _ _ _ _ _ _ (1pt) What is odd about your result?:____________________________________________ (1pt) What did you have to do in order to find the consensus? _______________________ (be specific)

Q2: You set up the following one-hybrid screening system using the consensus sequence : Draw an arrow as to where you would place your consensus sequence In the plasmid below. Is there a benefit to putting more than 1 site (1pt)? _____________________________________________________________ What does the red box represent in this experiment (1pt)?___________________________ (be specific) Plasmid above has a minimal promoter (TATA box) Q3: Using the 1000 different cDNAs from Q1, please describe what you will fuse them to and why?(2pts)