Agarose gel electrophoresis

Agarose gel electrophoresis

Aim

To perform electrophoretic separation of given DNA using agarose gel electrophoresis

Principle

The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biological molecules such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations (+) or anions (-). Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

The equipment required for electrophoresis consists basically of two items, a power pack and an electrophoresis unit. Electrophoresis units are available for running either vertical or horizontal gel systems. Agarose gels for DNA electrophoresis are run submerged in the buffer in horizontal gel systems. The power pack supplies a direct current between the electrodes in the electrophoresis unit.

Agarose is a linear polysaccharide (average relative molecular mass about 12 000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations of between 1 % and 3 %. Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution is then poured into a mould and allowed to solidify. As it cools, agarose undergoes polymerization i.e., sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which is determined by concentration of agarose.

Agarose gel electrophoresis is used to separate DNA or RNA molecules by size. Nucleic acids are negatively charged and are moved through an agarose matrix by an electric field (electrophoresis). Shorter molecules move faster and migrate further.  Agarose gel electrophoresis is one of several physical methods for determining the size of DNA.  In agarose gel electrophoresis, DNA is forced to migrate through a highly cross-linked agarose matrix in response to an electric current.  In solution, the phosphates on the DNA are negatively charged, and the molecule will therefore migrate to the positive pole.  There are three factors that affect migration rate through a gel; size of the DNA, conformation of the DNA, and ionic strength of the running buffer.  

Materials required

1. Horizontal electrophoresis system with power pack

2. Transilluminator

3. Agarose

4. Ethidium bromide

5. Loading dye (0.25% bromophenol blue in 50% glycerol)

6. 10X TBE buffer (0.89 M Tris, 0.89M Boric acid and 2 mM EDTA, pH - 8)

Procedure

1. A clean dry gel casting tray was prepared by sealing the open edges with cellotape.

2. 50 ml of 0.8% agarose solution in TBE buffer was melted completely into a clear solution. The molten solution was cooled to 50^oC and 10 µl of ethidium bromide (10mg/ml) was added, mixed well and poured in to the casting tray.

3. A well forming comb was placed at 1 cm from one end of the plate so that the teeth of the comb did not touch the bottom of the plate.

4. The comb was carefully removed from the agarose matrix once it is solidified and the gel along the casting tray was placed on the electrophoresis tank after removing the cellotape.

5. The tank was filled with 1X TBE buffer until the gel is submerged.

6. The gel was loaded with DNA samples (15 µl of DNA + 5 µl of loading dye)

7. The electrophoresis unit was connected to electrodes of the power supply unit so that cathode comes near to the DNA loaded end.  The voltage was set to 100 V and allowed to run for 2 hours

8. The run was terminated when the fast running bromo phenol blue crosses two thirds of the gel.

9. The bands of DNA were visualized on UV transilluminator.

Result

The bands of DNA were visualized on UV transilluminator.