Question on \"Plasmid conformations and mobility on agarose gel vs. oxidative st

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Question on "Plasmid conformations and mobility on agarose gel vs. oxidative stress"

Extra resource on this subject: http://www3.interscience.wiley.com/cgi-bin/fulltext/113449269/PDFSTART

a) Summarize plasmid conformations and mobility on agarose gel vs. oxidative stress.

b) How could you assess if a plasmid was completely digested by a restriction enzyme or not by exclusively considering the results from agarose gel electrophoresis?

c) Could you estimate the size of a supercoiled plasmid using the MassRuler Express Forward DNA ladder?

Explanation / Answer

a) Plasmids exist in three conformations: Supercoiled, Circular and Linear.Under normal conditions, a large fraction of plasmids exist in supercoiled form. On an agarose gel at constant voltage, the supercoiled plasmid which has the most compact conformation runs lowest, followed by linear and then circular which has the most spread out conformation and hence moves slowest through the pores on the agarose gel.

As seen in the paper, oxidative stress conferred by free radical ions (sourced in this case from SnCl2) cause bulges and breaks in the DNA. In supercoiled DNA, creating nicks relieves tension of the supercoils and loosens the compact conformation. With an increase in SnCl concentration we are hence able to see the increase in linear and circular forms of plasmid due to double and single stranded breaks respectively.

b) complete digestion by restriction enzyme would mean that the entirety of the plasmid on the gel would run at the linear size as a single sharp band.

c) It is possible to arrive at an ESTIMATE using the MassRuler Express Forward DNA ladder, since the ladder is used to measure short size differences and the bands run close to each other.

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