Agarose Gel Electrophoresis

Agarose Gel Electrophoresis Experiment Theoretical Background

•  Gel Electrophoresis is a procedure used in molecular biology to separate and identify molecules (such as DNA and RNA) by size. The separation of these molecules is achieved by placing them in a gel made up of small pores and setting an electric field across the gel. 
• The molecules will move based on their inherent electric charge (i.e., negatively charged molecules move away from the negative pole) and smaller molecules will move faster than larger molecules; thus, a size separation is achieved within the pool of molecules running through the gel. The gel works in a similar manner to a sieve separating particles by size; the electrophoresis works to move the particles, using their inherent electric charge, through the sieve.
• This principle is easily explored using virtual lab gel electrophoresis simulation. 

Agarose Gel Electrophoresis Principle of Work

•  Agarose is isolated from the seaweed genera Gelidium and Gracilaria. It’s mixed with a buffer and heated in a microwave, then left to cool down before pouring in the cast. A comb is added at a specific site to form the wells required for sample upload. Then the gel is left to solidify. 

The steps can be practiced using an agarose gel simulator online.

• The concentration of gel = weight of agarose/volume of buffer (g/ml). For a standard agarose gel electrophoresis, 0.8% gel gives good separation of large 5–10kb DNA fragments, while 2% gel gives a good resolution for small 0.2-1 Kb DNA fragments.

• During gelation, agarose polymers associate non-covalently and form a network whose pore sizes determine a gel's molecular sieving properties.

• The phosphate in the backbone of DNA is negatively charged, therefore DNA fragments will migrate to the positively charged anode.
• DNA has a uniform mass/charge ratio, therefore DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight.
• The rate of migration of a DNA molecule through a gel is determined by the following: 

1. Size of DNA band (the heavier, the slower)

2. Agarose gel concentration (usually 0.8%).

3. DNA conformation (linear/plasmid/etc..).

4. Voltage

5. Electrophoresis buffer.

6. Ethidium bromide: EtBr is positively charged, thus; reduces the DNA migration rate by 15%. Other stains for DNA in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blue.

A DNA ladder is a mixture of DNA molecules of known lengths that is used as a reference to estimate molecular sizes of unknown DNA fragments, during a gel electrophoresis online simulation.

• UV light activates electrons in the aromatic ring of ethidium bromide releasing light as electrons return to ground state. EtBr intercalates itself in the DNA molecule in a concentration dependent manner. So higher intensity means higher amount of DNA.
• The agarose gel electrophoresis experiment is useful in pcr product analysis, when visualized in virtual lab gel electrophoresis