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Thread: Recombinant DNA Questions

  1. #1 Recombinant DNA Questions 
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    I am a immunology major entering my junior year with a very small biology background. I am struggling understanding the following questions:
    Can you please help me understand the reasoning behind these questions and how to understand the concept. Any advice, urls, would be appreciated. This experiment was cloning a kanamycin resistance gene into a plasmid and determining the orientation of insert.


    1. Which of the following pairs could be ligated together? (All ter- mini are cohesive and complementary.)
    a. 5'-dephosphorylated linear insert DNA + linear vector
    b. Supercoiled vector + linear insert DNA
    c. 5'-dephosphorylated linear vector + linear
    5'-dephosphorylated insert DNA
    d. Linear 5'-dephosphorylated vector + linear insert DNA
    e. Nicked vector + linear insert DNA
    In general, which of the above possibilities would be the best ap- proach in a subcloning experiment like the one you have done? Why?

    2. Assume the transformants produced with the ligated DNA were also plated on ampicillin medium. Would you expect to see a significant difference in the number of colonies compared to the kanamycin plates? Why? (Hint: the linear vector was not dephosphorylated before the ligation).

    2b. Why would it be unwise to pick a transformant from an ampicillin plate if you were trying to isolate the recombinant DNA? If you had, is there a step in this series of experiments that would have prevented the propagation of the incorrect plasmid?

    3. Did the electrophoretic pattern of your uncut recombinant plasmid contain many forms of DNA like your ligation reaction? Explain.


    4. Can the size of a supercoiled plasmid be calculated by compari- son to linear DNA fragments of known size that have been run in parallel?

    5. A Kanr transformant was found to contain the supercoiled pUC vector without an insert in addition to the expected supercoiled recombinant plasmid. How can this be explained?

    Thanks in advance.


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  3. #2  
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    If you're doing work set for a course, you really need to do some work yourself before asking us for help.

    We can't read your mind about what causes you difficulty.

    And we're not here to do your work for you.


    Have you tried any searches for the information yourself?

    Does the whole topic give you general conceptual problems or is it the specifics that trip you up?

    Do you have a textbook or lecture notes that are supposed to help or is this stuff beyond the scope of any such texts?

    Try out some draft answers for yourself and post them for advice about whether you're on the right track.

    (I won't be able to help you. But someone else will, I'm sure.)

    And I'll shift this to Biology.


    "Courage is what it takes to stand up and speak; courage is also what it takes to sit down and listen." Winston Churchill
    "nature is like a game of Jenga; you never know which brick you pull out will cause the whole stack to collapse" Lucy Cooke
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