rct_ans_3_16

rct_ans_3_16 - MIT Department of Biology 7.28, Spring 2005...

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MIT Department of Biology 7.28, Spring 2005 - Molecular Biology Question 1. 1A. You are studying the function of RecA in vitro along with a lab partner. You run several reactions to examine the requirements for RecA activity. For each reaction below give the expected structure of the DNA products after RecA has been removed (by phenol extraction.) Regions of homology are shown as thick lines . Radiolabelled DNA strands are indicated by the *. * + RecA ATP 1) 4 kb ssDNA 4 kb dsDNA * + RecA ATP * + 4 kb ssDNA 4 kb dsDNA * + Reaction lacks a topoisomerase or a free DNA end in the region of homology so DNA strands cannot intertwine. Reaction lacks a region of 2) ssDNA for RecA binding . 4 kb dsDNA 4 kb dsDNA 4 kb dsDNA RecA * 3) + ATP gyrase * * * RecA * Lacks a region of homology 4) + + for RecA-dependent strand ATP invasion. 4 kb ssDNA 4 kb dsDNA 5) * RecA + + 4 kb dsDNA ATP * 4 kb dsDNA 4 kb ssDNA
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1B. After you carry out these reactions, your partner runs a non-denaturing acrylamide gel to analyze the products. For each reaction, she runs 3 lanes. Lane (a) is a control reaction run without RecA. Lane (b) is the complete reaction loaded directly into the gel. Lane (c) is the complete reaction that has been phenol-extracted to remove protein prior to loading on the gel. She exposes the resulting 15-lane gel to X-ray film. She has to leave at this point, and after developing the film, you sit down to write up the lab report. Unfortunately, you realize that you do not know what order she loaded the reactions in.
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rct_ans_3_16 - MIT Department of Biology 7.28, Spring 2005...

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