There are several physical methods available for separating nucleic acid (DNA and RNA) based on its size. Gel electrophoresis is a separation technique which is purely based on charge and size. Agarose gel electrophoresis is eventually one of the traditional methods of separating and analyzing nucleic acid. In this method, gel made from agarose acts as a separating medium. Agarose which is linear polymer agarose is a polysaccharide, whose monomeric unit is a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose.

Agarose is in powdered form, and is insoluble in water at room temperature. It gets dissolved in boiling water and when it starts to cool, it undergoes cross-linking (H-bonding) and results in polymerization (agarose gel matrix). Extent of cross-linking depends on percentage of agarose (higher percentage results in higher cross linking thus more sieving effect due to small pore size).

As discussed early in the lecture, the charge of the DNA is negative and therefore it migrates to the anode (positively charged electrode), if a voltage is applied. Nucleic acid molecules are separated by applying an electric field to move the DNA through an agarose gel matrix. The migration rate of the DNA is mainly affected by the factors such as size of the DNA, agarose concentration used and conformation of the DNA.

The migration of the molecules in gel electrophoresis is directly proportional to the size of the molecule. The gel sieves the movement of the molecules based on their size. Small molecules migrate faster and than bigger ones as small molecules can move more easily through gel pores. Due to difference in the migration rate of various size DNA molecules in gel DNA fragments are separated based on sizes. The size of the fragments can be determined by running standard DNA ladder run in parallel. The relationship of migration rate to size is linear throughout most of the gel, except for very largest fragments. The fragments such as genomic DNA which are very big in size shows difficulty in penetration through the gel pores. Hence, does not show linearity. The precise relationship between migration rate and size can be calculated as rate is inversely proportional to the log₁₀ of the number of base pairs in a DNA fragment.