Intact, circular plasmids don't run true-to-size. Both positive and negative supercoiling can cause plasmids to look larger or smaller than expected on a gel. The easy fix it to find a single cutter and linearize the plasmid.

But a difference that large means that you might be mistaken about the size (or identity) of the control plasmid.

Double-check your notes and the band sizes in your DNA ladder.

Good luck!

Hello Eugenia,

I agree with Katie that the supercoiled form of a plasmid generally migrates differently from the linearized form. Most of the time in my hands it migrates faster probably because it forms a more compact species due to the supercoils but the migration rate is sensitive to the conditions (temperature, etc...). In general it also gives a wider and fuzzier band because of all the differently supercoiled species present. In my hands however, nicked plasmids migrate like the linearized form. Another possibility is that you have a plasmid dimer, which happens by homologous recombination between copies of the same plasmid. You end up with a plasmid twice the size of the initial one (if the starting plasmid has genes ABC in that order the sequence of the dimer will be ABCABC), but for many purposes it does not change anything: when you cut with a "one-cutter" enzyme, it will have two sites in the dimer and generate the same linearized form as the monomer. When you do a PCR each primer anneals in two places in the dimer, but you have probably used an elongation time corresponding to the monomer so you get mainly the monomeric linear form.

pWhitescript plasmid was supplied with the kit of agilent and I searched it, but I could not find something helpful. Probably, I will try it with a different control. Thank you very much for your answer. It was very helpful!!