Biotechnology and Bioinformatics (1+1)

Agarose Gel Electrophoresis

Agarose forms a gel by hydrogen bonding and the gel pore size depends on the agarose concentration. The DNA molecules are separated by electrophoresis on the basis of their size, shape and the magnitude of net charge on the molecules. When an electrical field is applied across the gel, DNA which is negatively charged at neutral pH, migrates towards, the anode. Larger molecules migrate more slowly through the pores of the gel less efficiently than smaller molecules. A linear DNA fragment of a given size migrates at different rates though gel containing different concentration of agarose. There is a linear relationship between the electrophoretic mobility of DNA and the gel concentration. Thus, by using gels of different concentrations, it is possible to resolve a wide size range of DNA molecules.

Agarose gel has lower resolving power than polyacrylamide gel but have greater range of separation of DNAs from 200 bp to 50 kb in targets can be separated. Agarose gel is run horizontally and can be fragile and difficult to handle safely. When preparing very concentrated (4% or greater) gels the agarose should gradually be added to a vigorously stirred buffer to avoid clumping. Should finer resolution be necessary, polyacrylamide or sequencing gels can be used. Approximately a quarter of the PCR product should be sufficient to be seen directly on the ethidium bromide stained gel.

Ethidium bromide can be added to the gel to a concentration of 1 m g/ml, or gels can be stained after electrophoresis. The dye ethidium bromide intercalates between the bases of RNA and DNA and fluoresces orange when irradiated with UV light. Polyacrylamide gels are effective for separating small fragments of DNA (5-500 bp), their resolving power is extremely high, and fragments of DNA that differ in size by little as 1 bp can be separated from one another. The disadvantage is that more difficult to prepare and handle.