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.
For more information please visit the the following link:
