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How do you tell the difference between linear, relaxed circular & supercoiled form of pUC18 on an agarose gel?
I know in an agarose gel, the smallest, lightest molecules travel the farthest, hence linear would make it the farthest. However, why does the relaxed circular DNA travel the least and supercoiled travel the 2nd least?
Thanks for any help!
Because the supercoiled is twisted up on itself, making it more compact so it can move through the gel a bit easier than the relaxed circle.
Also, the linear doesn’t always travel the fastest. Depending on the agarose percentage, the buffer you’re using, and the voltage on the gel, some times the linear will migrate in between the open circular and the supercoiled. Best way to be sure which band is linear is to compare with a sample that’s been linearized with a restriction enzyme known to cut only once.
Shingo Nakamura – Linear Light (Original Mix)
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