Final2006 - Take-home Final Bio 325/807 Spring 2006...

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Take-home Final Bio 325/807 Spring 2006 Take-home final exam that is due in my office by noon on Friday April 28, 2006 . You may hand it in earlier if you wish. You are expected to consult your laboratory manual, the reference book Molecular Cloning by J. Sambrook (in library) or any other reference book or web site. For some questions there is no one correct answer, you will be graded on throughtfulness. 1. When subcloning a DNA fragment into the plasmid, pUC18, transformants are plated on agar plates containing ampicillin and X-gal. Why are amp and X-gal added? pUC18 plasmids contain a polylinker (or multiple cloning site) for cutting the plasmid with restriction enzymes to insert foreign DNA. What is the advantage of using two different enzymes for subcloning, such as BamHI and HindIII as we did, as opposed to using a single enzyme? If you did design a cloning strategy that used only a single enzyme, how would you determine the orientation of the fragment in pUC18? 2. There are two common methods used to isolate a sub-portion of DNA from a larger DNA piece, such as starting from genomic DNA. One is to use restriction enzymes to cut the fragment into smaller pieces and the second is to use PCR (polymerase chain reaction). a. What three steps occur during PCR? b. Draw a diagram showing how the selection of specific PCR primers determines the region of DNA that is amplified. c. If I start with 10 ng of DNA template, how much DNA will I have generated after 30 cycles, assuming no reagent became rate limiting. d. Can PCR be used to amplify an RNA template? If yes, how is this accomplished? PCR can be used to amplify an RNA template through the Reverse Transcription PCR (RT-PCR). RNA is first reverse transcribed into its complementary DNA, then it is amplified.
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3. Using genomic DNA isolated from our cheek cells, we used primers specific to the human TPA (Tissue Plasminogen Activator) gene to amplify fragments of either 100 or 400 bp. a. What is responsible for the length polymorphism in the 25
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This note was uploaded on 11/16/2010 for the course BIO 234 taught by Professor Palter during the Spring '10 term at Temple.

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Final2006 - Take-home Final Bio 325/807 Spring 2006...

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