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PS1KEY

# PS1KEY - Professor Hinck Bio 110 problem set 1 1 In...

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Professor Hinck Bio 110, problem set 1 1) In velocity sedimentation experiments, tropomyosin, at 93kd sediments at 2.6 S, whereas the 65kd protein hemoglobin sediments at 4.3 S. Remember: the sedimentation coefficient is a linear measure of the rate of sedimentation. The a-carbon backbone models of these 2 proteins are shown. How is it that the bigger protein sediments slower than the smaller one. The rate of sedimentation of a protein is based on size AND shape. The nearly spherical hemoglobin will sediment faster than the more rod-shaped tropomyosin, even though tropomyosin is a larger protein. A spherical protein with its smaller surface-to-volume ratio will experience less drag than a rod, and therefore sediments faster. Because most proteins (although not hemoglobin and tropomyosin) have about the same density (mass/unit volume), velocity sedimentation is preferred to equilibrium centrifugation for the separation of proteins. Equilibrium sedimentation is commonly used to separate organelles, which have different densities. 2) Imagine that you are working for the Centers for Disease Control and that you are in the Amazon basin trying to identify the causative agent of a newly discovered infectious disease. You suspect that the disease is caused by bacteria. What would be the most simple and direct procedure that you could use to determine if the blood of infected individuals contains bacteria? Explain your answer. Explain your answer. (4 points) I would use the polymerase chain reaction (PCR) using primers designed to amplify the 16S ribosomal RNA gene and compare this sequence with that of known bacteria, archaea, or eukaryotes. Another answer worth points would be microscopy but this would not be as accurate of a procedure 3) You want to amplify the DNA between the two stretches of sequence shown below. Of the listed primers choose a pair that will allow you to amplify the DNA by PCR.

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Professor Hinck Bio 110, problem set 1 ANS) The appropriate PCR primers are primer 1 (5’-GACCTGTCCAAGC) and primer 8 (5’-TCAATCCCGTATG). Even though primer 1 doesn’t match exactly (2Cs instead of 2Gs), the rest of the matches will allow annealing and it will hybridize to the bottom strand and prime synthesis in the rightward direction. The second primer will hybridize to the top strand and prime synthesis in the leftward direction. (Remember that strands are pair antiparallel.) Note that primers 2,3,6,& 7 won’t hybridze to either strand. The remaining primers 4 & 5 will hybridize, but prime synthesis in the wrong direction. 4) You have raised four different monoclonal antibodies to Xenopus Orc1, which is a component of the DNA replication origin recognition complex (ORC) found in eukaryotes. You want to use the antibodies to immunopurify other members of ORC. To decide which of the monoclonal antibodies ---TK1, TK15, TK37 or TK47--- is best suited for this purpose, you perform an immunoprecipitation assay. To do this, you covalently link the antibodies to beads, incubate them with Xenopus
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