Quantitation of ethidium-stained closed circular DNA in agarose gels

The fluorescence of ethidium bromide (EB) bound to equimolar amounts of supercoiled form I and unstrained linear form III pBR322, SV40 and PM2 DNA in agarose gels has been measured by scanning a photographic negative of the gel with a microdensitometer. For SV40 and PM2 DNA, commonly used staining conditions cause both forms, i.e. linear and supercoiled, to fluoresce to the same extent. This obviates the need to use a correction factor for the fluorescence of form I DNA when measuring the amount of this form relative to the amounts of unstrained forms in agarose gels. In the case of PBR322 DNA, form I was found to fluoresce approximately 20% more than form III DNA.