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Agarose gel of a single-cut plasmid and the same plasmid without treatment

Hi, in this agarose gel photo (70 volts, 3.5uL Gel Red in 30ml of TBE, 1% Agarose, 1 hour run), I've checked the digestion of a large plasmid (15Kbp, one cut site). The theory says that the digested plasmid (linearized) should migrate slower than the supercoiled form of the uncut one (bigger band of the Undigested Plasmid). In my case the digested plasmid is faster than the undigested one. I've found that my digested plasmid could be single-stranded (http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/). Do you think that could be the case?

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You assume that the undigested plasmid is supercoiled, but more probably it is in the relaxed circular form due to single strand nicks. This form migrates slower than linear DNA.

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I think that the simplest explanation of what you are seeing is that the uncut plasmid is in the form of dimers formed by recombination between monomeric circles. These would be resolved into monomer-sized linear molecules in the digestion.

I haven't seen this idea of single-stranded circles before, but if it did happen then I don't think this form would be predominant, and of course such ssDNA circles would be unaffected by digestion.

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  • $\begingroup$ In my opinion the undigested plasmid is ok, maybe during the digestion or after the plasmid became single-stranded. $\endgroup$ – J.G. Jul 15 '17 at 12:49
  • $\begingroup$ Why would the plasmid spontaneously convert from dsDNA to ssDNA, in your opinion? $\endgroup$ – Alan Boyd Jul 15 '17 at 13:18
  • $\begingroup$ I don't think that the plasmid spontaneously convert to ssDNA, but maybe I do something wrong, maybe something like pipetting too vigoursly? I don't know. My tutor says the band in the gel must be higher. I've done the cut 3-4 different times, also with a different enzyme but always the same result. $\endgroup$ – J.G. Jul 15 '17 at 14:24

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