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Bi1_2011_PS4 - Bi1 The Biology and Biophysics of Viruses...

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Bi1: The Biology and Biophysics of Viruses Spring 2011 Problem Set 4: Genomics and Cloning Due Tuesday, April 26 at 4:00 P.M. in the Bi 1 closet Name: ______________________________________________________________ Section # : ____________________________________________________________ Mail Code : ___________________________________________________________ TA Names : ___________________________________________________________ Date and Time turned in : _______________________________________________ Number of pages including this one : ______________________________________ AFTER YOU FINISH: How long did it take you to complete this problem set? ____________________________ Go to the Bi1 moodle site at http://courses.caltech.edu/ and take the homework survey There are 2 questions. The number of parts to each question is listed at the beginning of each; be sure to answer all the parts! Grade: Problem 1 __________ Problem 2 __________ TOTAL: _________ HOMEWORK INSTRUCTIONS 1) Turn in your homework stapled to this cover page. 2) Use separate sheets of paper for your answers. 3) Write or type your answers neatly. 4) Put your name on each page of your answers. 5) Box your answers, please, so that the grader can find them. Points may be deducted if you don’t follow these instructions!
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Problem 1: Restriction enzymes (50 points – 6 parts) Readings: Ch16, Ch19 (pp. 391-395), and B12-13 A common feature of plasmids used in molecular cloning is a short segment of DNA called a “multiple cloning site” (MCS), which is used to allow easy insertion of a new DNA sequence into the plasmid. A multiple cloning site contains many unique (i.e., occurring only once) restriction sites. You decide to clone (i.e., insert) your favorite gene into the pUC18 plasmid for expression of the gene product in E. coli . To insert your gene, you have designed two PCR primers that are complementary to the 5' and 3' ends of your gene, respectively. Each primer also includes a restriction site (chosen from among the sites that are in the MCS of pUC18). Once you have amplified your gene via PCR, you mix it with a single restriction enzyme (if the restriction sites are the same on both ends) or with two restriction enzymes (if the sites are different) in what is called a “digestion” reaction. You also digest the pUC18 plasmid with the same restriction enzyme(s), to produce compatible ends for your PCR fragment. You incubate the cut plasmid and PCR fragment together with an enzyme called DNA ligase. Ligase reseals the breaks after the overhangs anneal, so that the PCR fragment becomes covalently linked to the plasmid DNA. (The ligation reaction is not 100% efficient, so you end up with some plasmids that do not contain inserts.) The ligase reaction mixture is then transformed (inserted) into E. coli , and individual colonies are screened to find those containing a plasmid with an insert. Below is a (simplified) map of the pUC18 plasmid. Note that the right of the map shows a magnified
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Bi1_2011_PS4 - Bi1 The Biology and Biophysics of Viruses...

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