Last week I tried to introduce single-strand nicks into a closed circular plasmid (pUSS-R) by doing restriction digestion in the presence of ethidium bromide (background is here). I tested two different dilutions of HindIII, a restriction enzyme that should cut this plasmid once, with three different concentrations of ethidium bromide, to find conditions where a convenient range of incubation times gave a range of partially digested molecules.
I was hoping to see what's shown in the upper gel drawing - appearance of a novel band that migrated slower than the fully digested DNA in the rightmost lane and much slower than the supercoiled DNA in the leftmost lane. This new band would contain plasmid that had undergone a single-strand nick at its single HindIII site. I didn't know whether I might also see eventual appearance of linear DNA. But instead I saw what's shown in the middle gel drawing - gradual appearance of a linear-sized band as the supercoiled band disappeared.
I wondered if the HindIII now being sold no longer causes nicking (maybe New England Biolabs has 'improved' their HindIII clone...). So I did the control shown in the lower gel drawing, incubating pUSS-R with a very low concentration of DNase I, an enzyme widely used to create nicks in double-stranded DNA. Again I expected to see a novel, slow-migrating band, but instead saw only a linear-sized band. Damn!
I also ran samples from a few old plasmid preps. Most of these contained several bands, but I don't know if the slowest one is relaxed circles or dimers, because the plasmids may have been prepared from rec+ cells. I've now also checked the old literature, just in case I was mistaken in expecting nicked/relaxed DNA ("form II") to migrate slowly, but indeed it should (see for example the marker lanes in Fig. 2 on PNAS 86:1309).
Now what? Is the problem that the nicked DNA migrates at the same speed as linear DNA? Is this specific to this particular plasmid? Or do I not have any nicked DNA? Should I try another plasmid?