DNA fragments of length ranging from 50 base pair to several million base pair can be separated using agarose gel electrophoresis. Migration rate of the fragments also depends on the concentration of agarose used to prepare gel. The concentration of agarose is inversely proportional to the rate of migration of the DNA fragments, i.e. lower the concentration, the faster is the DNA migration rate and vice versa. Generally used agarose concentration is 0.7% to separate DNA fragments of range 2- 10 kb and 2% agarose for separation of small fragments such as 0.1- 1 kb. Low percent gels are weak and high percent gels are often brittle. Standard 1 % agarose gels are common for many applications, which can resolve DNA fragments from 0.5- 30 kb in length.

The above explained relationship between migration rate and size of DNA is applicable only to linear DNA fragments. Generally DNA can exist in three forms: linear form, opencircular form and supercoiled form. The linear DNA may be the product of PCR amplification or the restriction digestion product. But plasmid DNA is the one which are mostly studied. In in-vivo plasmids exist as highly supercoiled form to enable it to fit inside the cell. When the plasmid preparation is done plasmid DNA can exist in all the three conformations i.e. linear, opencircular and supercoiled forms. In a good careful plasmid preparation, most of the DNA will remain supercoiled along with the opencircular and linear form. The opencircular form of plasmid exist due to the break in only one of the strands, the DNA will exist as circular. The break will allow rotation around the phosphodiester back bone of DNA due to which supercoils will be released. The plasmid exists in the linear form when there is a cut in the circular DNA. The supercoiled form of plasmid DNA sustains less friction when run in agarose gel matrix. Therefore, for the same size plasmid DNA, supercoiled DNA form will runs faster than open circular form and linear form. When a pool of plasmid sample containing supercoiled, opencircular and linear form is run on agarose gel, supercoiled DNA will run faster which is the bottom most band, next is the nicked form i.e. opencircular form which is the middle band and the top most band in the gel is the linear or relaxed form (Fig. 4). In unicellular, plasmids tend to recombined with themselves to produce smaller multimers. Therefore, an uncut plasmid produces a complex banding pattern on an agarose gel. When the plasmid is cut with a single restriction enzyme, the super coiled, open circular and multimer forms are reduced to a linear conformation. The amount of different conformation of the plasmid depends on number of manipulations that are incurred in plasmid preparation. If the plasmid preparation is not good linearization of supercoiled DNA may occur because of the nick produced in the plasmid in preparation process. Due to which in a plasmid preparation, the amount of supercoiled DNA may be small amount to nil, which can be clearly seen on agarose gels. Some times improper handling or storage of the isolated DNA may degrade, which can be detected as a smear when run on the agarose gel.