I am currently working with a plasmid with an insert separately mutated in 3 diffferent locations (giving 1 unmutated and 3 different mutated variants). When I run the undigested plasmids (4.5 kB with insert) on an 0.8% agarose gel the unmutated variant and 2 mutated variants separate identically with 1 supercoiled and 1 relaxed band each. However, the third mutated variant produces similar bands but way further up in the gel. According to the ladder these bands are twice as big as for the three other variants (I know size is difficult to judge with intact plasmids, but still..). When I digest the plasmids with single-cutters XhoI and HindIII from Fermentas I see single bands with the same size for all plasmid variants (and an extra small band for the insert when cutting with both RE simultaneously). So my question is - can the migration pattern for undigisted plasmids vary this much (dispite exactly the same bp lenght of the plasmids and only a few bp variance in sequence)? Or can the different migration be due to dimerization of my plasmid?
Super if someone could give me some input on this!