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A number of factors can affect the migration of nucleic acids: the dimension of the gel pores (gel concentration), size of DNA being electrophoresed, the voltage used, the ionic strength of the buffer, and the concentration of intercalating dye such as ethidium bromide if used during electrophoresis.

Smaller molecules travel faster than larger molecules in gel, and double-stranded DNA moves at a rate that is inversely proportional to the log10 of the number of base pairs. This relationship however breaks down with very large DNA fragments, and separation of very large DNA fragments requires the use of pulsed field gel electrophoresis (PFGE), which applies alternating current from two different directions and the large DNA fragments are separated as they reorient themselves with the changing current.

For standard agarose gel electrophoresis, larger molecules are resolved better using a low concentration gel while smaller molecules separate better at high concentration gel. High concentrations gel however requires 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. 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.