AGAROSE GEL ELECTROPHORESIS 

 Agarose gel electrophoresis is a widely used technique in molecular biology to separate and visualize DNA fragments. The technique relies on the fact that DNA has a negative charge due to its phosphate backbone, and can therefore be separated by size as it migrates through a gel matrix under the influence of an electric field. Agarose gel electrophoresis is a powerful tool that allows researchers to visualize DNA fragments in a sample, assess the purity of DNA preparations, and confirm the success of DNA manipulation procedures such as PCR or cloning.

Principle of Agarose Gel Electrophoresis :

Agarose gel electrophoresis is based on the principles of molecular sieving and charge density. The agarose gel matrix consists of long chains of agarose molecules, which form a porous network of tunnels and spaces that can trap and separate molecules based on their size. The gel matrix is prepared by heating and dissolving agarose powder in a buffer solution, which is then poured into a casting tray and allowed to solidify into a flat slab or a column.

The electric field is applied to the gel matrix by placing two electrodes at opposite ends of the casting tray or column. The negatively charged nucleic acid molecules are attracted to the positively charged electrode and migrate through the gel matrix towards it. The migration rate is proportional to the size of the molecules, with smaller molecules moving faster than larger ones. The migration rate is also affected by the charge density of the molecules, with highly charged molecules moving slower than neutrally charged ones.

The separation of nucleic acid molecules in agarose gel electrophoresis is visualized by staining the gel with a dye that binds to the nucleic acid molecules and makes them visible under UV light. The stained bands represent the different sizes of the nucleic acid molecules that have migrated through the gel matrix.

Procedure of Agarose Gel Electrophoresis:

The agarose gel electrophoresis procedure involves several steps, including gel preparation, sample loading, electrophoresis, staining, and visualization.

Gel Preparation

The agarose gel matrix is prepared by dissolving agarose powder in a buffer solution, typically TAE or TBE, at a concentration of 0.5% to 2%. The agarose/buffer mixture is heated in a microwave or on a hot plate until the agarose is completely dissolved, and then cooled to a temperature of 50-60°C. The casting tray or column is prepared by applying a thin layer of petroleum jelly or agarose gel to the edges to prevent the gel from leaking out during the casting process.

The agarose/buffer mixture is poured into the casting tray or column and allowed to solidify for 20-30 minutes. The gel matrix is then removed from the casting tray or column and placed in an electrophoresis chamber filled with the same buffer solution used for gel preparation.

Sample Loading

The nucleic acid sample is prepared by mixing the DNA or RNA with a loading buffer, which contains a tracking dye and a density agent. The tracking dye allows visualization of the sample as it migrates through the gel matrix, while the density agent provides weight to the sample, enabling it to sink into the well.

The sample is loaded into the well of the agarose gel matrix using a micropipette. Care must be taken to avoid introducing air bubbles into the well, as they can interfere with the migration of the nucleic acid molecules.

Electrophoresis :

Electrophoresis is the process of separating charged molecules based on their size and charge using an electric field. In agarose gel electrophoresis, the charged molecules are nucleic acid molecules, such as DNA or RNA. The separation is achieved by placing the nucleic acid sample in a well in an agarose gel matrix and applying an electric field to the gel.

When an electric field is applied to the gel, the negatively charged nucleic acid molecules move towards the positive electrode. The migration rate is proportional to the size of the molecules, with smaller molecules moving faster than larger ones. The migration rate is also affected by the charge density of the molecules, with highly charged molecules moving slower than neutrally charged ones.

The agarose gel matrix acts as a molecular sieve, trapping the nucleic acid molecules as they migrate through the gel. The gel is composed of long chains of agarose molecules that form a porous network of tunnels and spaces that can trap and separate molecules based on their size. The gel matrix is prepared by heating and dissolving agarose powder in a buffer solution, which is then poured into a casting tray and allowed to solidify into a flat slab or a column.

The nucleic acid molecules are visualized by staining the gel with a dye that binds to the nucleic acid molecules and makes them visible under UV light. The stained bands represent the different sizes of the nucleic acid molecules that have migrated through the gel matrix.

Agarose gel electrophoresis is a commonly used technique in molecular biology for separating and analyzing nucleic acid molecules. It has a wide range of applications, including DNA fingerprinting, DNA sequencing, and gene expression analysis. The technique is relatively simple, cost-effective, and can be easily adapted to different experimental setups.