You cannot determine the size of covalently closed circular DNA by its electrophoretic migration, as this is dependent on the degree of supercoiling. Most likely, your Mw standards are linear DNA fragments, thus you should linearize your plasmids by digesting with an adequate restriction enzyme if you want to determine their sizes.

You cannot determine the size of covalently closed circular DNA by its electrophoretic migration, as this is dependent on the degree of supercoiling. Most likely, your Mw standards are linear DNA fragments, thus you should linearize your plasmids by digesting with an adequate restriction enzyme if you want to determine their sizes.

Pierre is correct, the only accurate way to really measure size is to linearize your plasmid by digestion with a restriction enzyme that only cuts a single time. Uncut plasmid DNA will often have many bands, the supercoiled monomer, the nicked or relaxed circle, and when present, multimeric forms of both of these. But when you cut the plasmid once, everything is reduced to a single band representing the size of the plasmid.

I agree with Pierre and Michael. Since your DNA is circular, you will get supercoiling, and this is going to cause your band to be “fuzzy”, and you may get bands a bit further down than the desire mark. Because supercoiled plasmids will migrate towards negative voltage much faster than the open circular Also, some of your DNA is may get knotted up, and this will cause it to move further than the DNA that isn’t knotted up. This can make a moderate to large blur form in your band. To increase accuracy, you generally want to cut open circular DNA before running it through a gel. I also attached a picture that will help you to understand migration of different DNA forms of plasmid in agarose.