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Agarose gel electrophoresis question


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#1 go100

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Posted 27 September 2010 - 04:05 AM

Hi all. General questions --
1. How can I assess if a plasmid was completely digested by a restriction enzyme or not by looking at the only the results from agarose gel electrophoresis?

2. Can you estimate the size of a supercoiled plasmid using the alphaquant1 DNA ladder?

-- In the lab, we used the restriction enzymes ECO RI and X hol to digest the amplicon from previous lab, as well as the destination plamid vector (pTrcHisB). This digest generates compatible sticky ends between pTrcHisB and the t7 RNA polymerase insert to form a recombinant plasmid (pTRcHisB/T7).

#2 philman

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Posted 27 September 2010 - 06:10 AM

1) Well it depends what you are digesting. Does the enzyme only have 1 cutting site in the plasmid? or multiple sites? If it only has one site then you can run digested and undigested DNA side by side, and the digested should be a single band on the gel, the undigested should have the multiple bands as typified by circular DNA.

It is always possible that there is some undigested DNA though, at concentration too low to see on Gel, I would reccomend digesting the the maximum time then cutting the band out of the gel and then gel-purifying the band to remove any contaminants.

2) I am unsure how accurate estimating size on a gel from a circular product would be. Would be more accurate to cut a small quantity of the DNA with an enzyme which only cuts once in the plasmid, then run that as a linear DNA on a gel next to the ladder, would give you a more accurate estimate of size.




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