Agarose Gel

  • Agarose gels are most commonly used for the size-based electrophoretic separation of nucleic acids (DNA and RNA). They can also be used for the separation of high molecular weight proteins or protein complexes including virus particles. In addition, fractionated biomolecules can also be eluted from agarose gel, thus allowing their purification.
  • The process of using agarose gel to separate or fractionate biomolecules under the influence of the electric field is called Agarose Gel Electrophoresis.
  • For moderate and large sized DNA ranging from ≈100 bp to ≈20 kilobase pairs, agarose gel electrophoresis is the most convenient and routinely used technique in most molecular biology laboratories (Stellwagen & Stellwagen, 2009)
  • As the name suggests, the agarose gel is prepared from agarose. Chemically, agarose is a polysaccharide, made up of alternating residues of 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-α- L-galactopyranose.
  • Agarose is almost insoluble in water. However, when suspension of agarose powder in an aqueous buffer is boiled, agarose melts and forms a viscous solution which upon cooling forms a stiff gel.
  • In contrast to acrylamide gel which forms through a process called polymerization, the process of the formation of agarose gel is called gelation. Due to this feature, agarose gels can also be melted and recast which is not possible with polyacrylamide gels.
  • Usually a slab gel, casted by pouring molten agarose in a mould, the casting tray, is used for the separation of DNA fragments by electrophoresis. This type of electrophoresis is called gel electrophoresis. Usually horizontal gel is casted for analysis of DNA and RNA.
  • Agarose gel pore size varies from 50 nm to 300 nm and can be adjusted simply by varying the concentration of agarose (denoted as percentage). Pore size of the agarose gel affects the resolution of DNA fragments. An optimal percentage of agarose Gel is required to achieve a good separation of a size range DNA fragments.
  • High percentage agarose gels are used for the separation of relatively small DNA fragments, whereas low percentage gels are good for the separation of large DNA fragments.
  • Agarose gel thickness and size (width and length) are controlled by the volume of agarose solution and size of the casting tray.
  • Not all agarose have the same melting and gelling temperature. Some agarose, called low melting agarose, is also available that is specifically used for the preparative purposes. Since agarose melts at low temperature, they allow purification without causing any harm to DNA fragments or protein complexes.

REFERENCES

  • Stellwagen & Stellwagen, 2009. Effect of the matrix on DNA electrophoretic mobility. J Chromatogr A. 1216(10), 1917-29. PMID-19100556; Full Text Link: sciencedirect, PMC (free download)

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