Analysis of nucleic acids by gel electrophoresis
The term electrophoresis describes the migration of charged particles under the influence of an electrical field. Many biological molecules such as amino acids, proteins, peptides, nucleotides and nucleic acid possess ionisable groups and therefore at any given pH exist in solution as electrically charged species either as cation or as anion. Under the influence of an electrical field these charged particles will migrate either to cathode or to anode depending on the nature of their net charges.
The equipment required for electrophoresis consists basically of two items a power pack and an electrophoresis unit. The power pack supplies a direct current between the electrodes in the electrophoresis unit. All electrophoresis is carried out in an appropriate buffer which is essential to maintain a constant state of ionization of the molecules being separated. When potential difference is applied across the electrodes it generates a potential gradient which is the applied voltage divided by the distance between the electrodes.
The factors affecting the rate of DNA migration in agarose gels are molecular size of DNA, agarose concentration, conformation of DNA, applied voltage, direction of electric field, presence of intercalating dyes and composition of the electrophoresis buffer.
|Agarose conc. in gel [% (W/v)]||Range of separation of linear DNA molecules (kb)|
|0.3||5 – 60|
|0.6||1 – 20|
|0.7||0.8 – 10|
|0.9||0.5 – 7|
|1.2||0.4 – 6|
|1.5||0.2 – 3|
|2.0||0.1 – 2|