Category: Agarose Gel Electrophoresis
Protocol: Size Determination of Plasmid DNA Using Agarose Gel Electrophoresis
Plasmids are circular DNA. Their size can not be determined in their native state as the migration of circular DNA in agarose gel is quite different and can not be compared with the linear fragments of ladder DNA. To know the approximate size of plasmid, the first step is to linearize the plasmid using a restriction enzyme that generates strictly a single cut. This results in a linear DNA that can be run on agarose gel and its size can be compared with the ladder sequence.
Applications of Agarose Gel Electrophoresis
Agarose gel electrophoresis is extensively used for the analysis of nucleic acid. Observations made by running DNA or RNA samples on agarose gel are very helpful to determine quantity and quality of nucleic acids. These powerful features make agarose gel electrophoresis an important essential step in many well known techniques such as northern and southern blotting, RFLP, RAPD, PCR, molecular cloning etc. Since agarose gel allows the elution of biomolecules, it can also be used for the purification of nucleic acids and large protein molecules and complexes. In addition, it can also be used to analyse biological processes such as apoptosis.
Agarose Gel Electrophoresis
Agarose gel, a porous matrix, acts as a sieve through which molecules can move. The electric field is the force that forces molecules to move through an agarose gel matrix. The molecules must have a net charge, positive or negative, in order to move in an electric field. A neutral molecule will not move in an electric field.