Techniques and Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis Introduction to agarose gel electrophoresis This technique uses an electrical current to pull DNA through an agarose based matrix. This is possible due to DNA having a net negative charge at physiological pH from its phosphate backbone Agarose is a high molecular weigh polysaccharide purified from seaweed that forms a porous matrix after it is forced into solution with heat and then allowed to cool down When electrophoresed through age, the smaller DNA molecules find less resistance to movement through the gel and move faster than larger molecules that are retarded by the polymer matrix Principle of separation by electrophoresis The basic principle of separation for all electrophoresis is the movement of a charged molecule in a medium that can be subjected to an electric field or… V is the velocity of the molecule subjected to electrophoresis; E is the electrical Eq field in volts/cm; q is the net charge on the molecule and F is the fractional V = f coefficient Composition of an agarose gel Once powdered agarose is hydrated and formed into a gel, it forms helical fibers and aggregates that create channels (also called pores) of 50 to over 200 nm in diameter The size of the pores in the solidified gel decreases as you increase the concentration of agarose in the gel Migration of DNA fragments through an agarose gel Larger pieces will be oriented closer to the wells on the gel, while smaller pieces will run closer to the bottom of the gel. Molecular weight markers (DNA ladder) can be run on the same gel as samples in order to estimate the size of any unknown DNA samples Visualization of DNA on an agarose gel DNA is not visible by eye on an agarose gel and it does not fluoresce when exposed to ultra violet light. Therefore, agarose gels must be stained to detect the DNA fragments Staining with ethidium bromide (EtBr) is a rapid, sensitive, and highly reliable method for visualizing DNA in gels. The stained gel is nominated from below with a short- or medium wavelength of UV light causing the EtBr, bound to DNA, to fluoresce brightly