Electrophoresis − the migration of a charged particle under the of an electric field.
Migration rate/separation depends on: size, shape, charge
The charge per unit length in any given fragment of DNA is the same since mobility of DNA molecules during gel electrophoresis
The small DNA fragments move more easily through the gel pores.
Requirements of the Gel electrophoresis
- Nucleic acid sample
- A supporting medium – agarose/polyacrylamide
- Buffer solution
- Electrophoresis apparatus – horizontal/vertical gel systems
- Staining agent (dye)
- Power pack
A supporting medium – agarose/polyacrylamide
Agarose is a linear polysaccharide made up, Both inter and intramolecular H-bond form a pores in the gel.
- Large pore sizes are formed by low concentrations
- Smaller pore sizes are formed by the higher concentrations
- Polyacrylamide gel is formed from the polymerization of the acrylamide monomers. The polymerisation is started by the addition of ammonium persulphate (APS) and the base tetramethylenediamine (TEMED)
- Acrylamide gels are defined in terms of the total percentage of acrylamide present
- Pore size in the gel can be changed by changing the concentrations of both the acrylamide and bis-acrylamide
Gel concentrations must be chosen to suit the size range of the molecules to be separated
Buffer Solution
Tris-borate-EDTA (TBE) – Tris base, boric acid and EDTA
Tris-acetate-EDTA (TAE) – Tris base, acetic acid and EDTA
Functions of the buffer solution.
- Provides electrolytes for pass through the current
- Reduce pH changes due the electric current
- Prevent any overheating of the gel
Staining agents
- Ethidium bromide – Binds to DNA by insertion between stacked base pairs, a strong orange/red fluorescence with UV light
- Silver staining –used for staining of nucleic acids in polyacrylamide gels
- Samples are prepared by dissolving them in a buffer solution, contains sucrose, glycerol, dyes (bromophenol blue)
