reorient themselves with the changing field. For standard agarose gel electrophoresis, larger molecules are resolved better using a low concentration gel while smaller molecules separate better at high concentration gel. Higher concentration gels, however, require longer run times (sometimes days). The movement of the DNA may be affected by the conformation of the DNA molecule, for example, supercoiled DNA usually moves faster than relaxed DNA because it is tightly coiled and hence more compact. In a normal plasmid DNA preparation, multiple forms of DNA may be present. Gel electrophoresis of the plasmids would normally show the negatively supercoiled form as the main band, while nicked DNA (open circular form) and the relaxed closed circular form appears as minor bands. The rate at which the various forms move however can change using different electrophoresis conditions, and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel. Ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence in gel during electrophoresis can affect its movement. For example, the positive charge of ethidium bromide can reduce the DNA movement by 15%. Agarose gel electrophoresis can be used to resolve circular DNA with different supercoiling topology. DNA damage due to increased cross-linking will also reduce electrophoretic DNA migration in a dose-dependent way. The rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. The resolution of large DNA fragments however is lower at high voltage. The mobility of DNA may also change in an unsteady field – in a field that is periodically reversed, the mobility of DNA of a particular size may drop significantly at a particular cycling frequency. This phenomenon can result in band inversion in field inversion gel electrophoresis (FIGE), whereby larger DNA fragments move faster than smaller ones. Migration anomalies "Smiley" gels - this edge effect is caused when the voltage applied is too high for the gel concentration used. Overloading of DNA - overloading of DNA slows down the migration of DNA fragments. Contamination - presence of impurities, such as salts or proteins can affect the movement of the DNA. Mechanism of migration and separation The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis.