When should I use ethidium bromide gel separations?
Ethidium bromide (EB) is used to both visualize DNA bands by fluorescence and when included in the running buffer to resolve circular from nicked DNA forms. Clearly in both cases, EB (at 0.5ug/ml) is required for detection of bands. The only difference is when it is used (either during electrophoresis or after). It is essential that, in either case, you destain with distilled water for 30 minutes prior to photography. You should include EB in the gel and buffer whenever you want to resolve nicked circular DNA (Open circular or OC DNA) from covalently closed circular (CCC) DNA (Panel A). Note that CCC DNA can either be relaxed or supercoiled. Without EB, two possible patterns are possible. As shown in Panel B, OC DNA and relaxed CCC DNA are sometimes poorly resolved and may migrate close together (see Panel B on the right). Panel C show another possible pattern. In this case, topo relaxation generates a series of topoisomers (circular DNAs that differ only in linking number) that migrat
