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Help Needed - Ligation Pairing (Vector and DNA)

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Help Needed - Ligation Pairing (Vector and DNA)

Postby techbrit » Sun Mar 13, 2011 7:34 pm

I am a first yr biotech grad student with no biology background. I am struggling understanding the question below
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?

Thank you so much for your help.
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