Principles of Gel Electrophoresis

Gel electrophoresis is a widely used laboratory method in biology to separate mixtures of macro-molecules such as DNA, RNA and proteins according to their molecular size. In this method electric current is used to push macro-molecule fragments loaded into slots made in the gel. The molecules to be separated migrate through pores in a thin layer of gel. Smaller the molecule, the greater distance it travels through the gel because the speed of the molecule is inversely proportional to the size of the molecule. 

Gel has two oppositely charged ends which create electric current. In this method, after loading the samples into the wells or slots, electrical current is passed through the gel. Since nucleic acids are negatively charged molecules because of their negatively charged sugar-phosphate backbone, the electric current repels the nucleic acid fragments through the gel towards the positive end of the gel. Different sized nucleic fragments travel at different speed and they form discrete bands that can be visualized using special stains.

Gel electrophoresis can also be used for separation of proteins due to their size so that they can be identified and studied. Proteins, however, are not negatively charged like nucleic acids so an extra step is required to make proteins negatively charged. Proteins are first mixed with a detergent solution, sodium dodecyl sulfate. This chemical unfolds proteins into almost a linear shape and covers them with a negative charge. After this step proteins can migrate through the gel by applying electric current.

Basics of Gel Electrophoresis Method

In this method the materials below are required to separate nucleic acids:

• Gel loading solution, 
• agarose,
• electrophoresis buffer 
• electrophoresis apparatus
• power supply 
• micropipette 
• stain

Gel loading solution helps samples to enter into the slots. It also includes a visible dye to monitor migration of the samples in the gel. Agarose is a polysaccharide and it is used to prepare the gel. It forms the separation matrix.  Electrophoresis buffer contains ions to create electrical current and it also keeps pH level same during the process. Electrophoresis apparatus holds the buffer solution and the gel.  Power supply produces the electric current to repel nucleic acids through the gel. Micro-pipette is required to transfer samples into the slots. Stain is used to visualize nucleic acids. Ethidium bromide is used as a dye for visualization of separated nucleic acids fragments. It goes between the bases stacked in the center of DNA helix. 

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Principles of Gel Electrophoresis