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As you might know there are 3 common forms of a plasmid:

  • ccc-form (CCC), being supercoiled.
  • oc-form (OC), being nicked and therefore relaxed.
  • linearized form (L), being cut on both strands and linear.

In theory the fastest migration should be the CCC, then L and the slowest is the OC-form. Source: https://www.sciencedirect.com/science/article/abs/pii/0147619X81900123

So I have a plasmid with 7631 bp from a Midiprep. I cut this plasmid with NotI and put both versions on a gel, to check for the quality of the Midiprep. I expected my plasmid to have the CCC form and therefore be faster (lower) than the cut L-form. I ran the gel and the result is the opposite. My plasmid runs slower (higher) than the cut version. Why is that? Do I have a misunderstanding somewhere? Is my plasmid ok?

enter image description here

For clarification: NotI- means that the plasmid is not cut. NotI+ means that the plasmid is cut (and therefore linear).

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  • $\begingroup$ What organism are you propagating your plasmids in? What kind of gel / buffer are you using? $\endgroup$
    – acvill
    Aug 24, 2023 at 13:46
  • $\begingroup$ E. Coli (DH5-alpha). This is a 1% agarose gel with 1x TAE. Digest was done with NEB enzymes and CutSmart-Buffer according to protocol. Non-cut plasmid was treated the same, but without enzyme. $\endgroup$
    – Felix H.
    Aug 24, 2023 at 13:54
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    $\begingroup$ @FelixH. Pred -> prep. If you are new at this game, you probably won't see much supercoiled, mostly circular or nicked circular. Supercoiled can also run in quite a few positions depending on the state and if you overloaded or not. I'm assuming that the NotI+ is running at the correct size (only 1 cut site)? If so, don't worry about how the uncut is running too much. That looks like 1kb plus ladder; if so, size is right. $\endgroup$
    – bob1
    Aug 24, 2023 at 21:27
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    $\begingroup$ It should have been "prep", a simple typo. Otherwise I agree with @bob1 $\endgroup$
    – Chris
    Aug 25, 2023 at 5:52
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    $\begingroup$ @FelixH. always good to challenge the paradigm! I agree your midiprep looks good. In my experience, supercoiled can run in a number of positions relative to cut; I can't recall exactly the reasons I was taught many years ago, but I think residual protein contamination, entangling of the ccc form with others, and overloading/underloading all play a role. $\endgroup$
    – bob1
    Aug 25, 2023 at 10:15

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