How can we justify the appearance of supercoiled plasmid

[Bhargav S]

DNA in agarose gel electrophoresis?

[waytoolongtobeawebname]

plasmids are protoviruses

[long_d0ng_s1lver]

Typical agarose gel electrophoresis is not a denaturing gel, so secondary structures will still be present. In denaturing gels, such as is needed for northern blots, denaturing agents are present either in the gel or loading buffer to linerarize the nucleic acids.

[Josh M]

As a first step, I would run the product on acrylamide or formaldehyde gel which would reduce secondary structure. If some seconary structure is broken down you would expect to see few one band or at least fewer bands on the gel compared to the original conditions. You could also run a gel at a higher temperature.

Also, adding Topoisomerase I or Topoisomerase II enzyme converts plasmid to a relaxed circular form. There are various commercial sources for the enzyme and previously described protocols.

[cheese]

It sounds like a lot of you are saying HOW to deal with supercoiled DNA in gel electrophoresis, but not WHY it is there. That is what I assume the question "how can we justify the appearance of super coiled plasmid DNA on a gel?" is asking.

I can think of three reasons, but I don't know how accurate they are.

1. Salt concentration of your reagents used in the miniprep could have an impact. For example, if your reagents were mixed wrong and the salt concentration is too high, your plasmid will supercoil. I don't feel comfortable just making up some kind of explanation for what kind of biochemistry is going on there.

2. Vector problem? I can't describe what, though.

3. Something in the bacterial growth medium?