Exam II 2007_Solutions

Exam II 2007_Solutions - SECOND EXAM CHE 339/BIO 335/BME...

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SECOND EXAM CHE 339/BIO 335/BME 339 FALL 2007 Thursday, November 8, 2007 WRITE IN PEN ONLY –IF YOU USE A PENCIL THERE WILL NOT BE A REGRADE! YOUR NAME _________________________________ 1. Library Generation (10 points) Briefly describe the steps in making a cDNA library. An antibody against the protein encoded by the desired gene is available. Explain how you can use this antibody to screen the library and isolate the gene of interest. Isolate mRNA from cells of interest by binding to a polydT column. Use reverse transcriptase to generate complementary DNA (cDNA). Treat with alkali to degrade RNA or RNAse. Synthesize complementary DNA strand using DNA polymerase and the base-paired 3’ hairpin as a primer. Treat with DNA Nuclease to cleave hairpin loop. Clone into vector that has been digesting with a blunt end restriction enzyme to generate compatible ends cDNA library. HANDOUT 11 AND 12 SEEM TO HAVE SLIGHTLY CONFLICTING INFO Plate bacteria on a Petri dish. Wait for bacteria to grow to form visible colonies. Transfer colonies to an induction plate using a filter. Make replica of colonies by pressing nitrocellulose filter onto dish. Incubate the filter with the antibody specific for the protein of interest. Wash filter. Incubate filter with radiolabeled secondary antibody. Develop using autoradiography and X-ray film. Use X-ray film to determine location of clones of interest. Pick clones, grow and sequence.
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2. Cloning and Expression Vectors (14 points total) A. What kind of vector would you use for the following purposes (1 points each): (i) Expression of a gene encoded by a 20,000 bp fragment in E.coli: Lamda phage (ii) Expression of a 4,000 bp fragment in yeast: Plasmid (iii) Expression of a gene for the production of a cellulose (a cellulose hydrolyzing enzyme) for the production of ethanol from cellulose in
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Exam II 2007_Solutions - SECOND EXAM CHE 339/BIO 335/BME...

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