Purpose: Electrophoresis is a method used to separate DNA molecules by sized restrictions are the enzymes used to prepare DNA for analysis or other manipulations. Here we examine the genotypic analysis of DNA using restriction enzymes and gel electrophoresis.
The various endonucleases used areECORI, Bain HI, and Hind III.Each of these enzymes has at least five or more restriction sites on the chromosome and it consequently produces six or more restriction fragments of different lengths.
The DNA strands are then immersed in a well of 0.8% agarose gel, which is the chosen medium.An electrical field, is applied to the gel evenly which makes the DNA fragments move faster from their original placement towards the positively charged electrode. The smaller DNA fragments travel faster through the gel than the larger ones. The gel actually acts like a sieve. Depending on the restriction enzyme used the DNA fragments separate into distinct bands during electrophoresis. The bands are then stained with a dye to make the invidual restriction enzymes evidently distinguishing them with a dye that attaches itself to the DNA molecule.
Electrophoresing can be detected by hydrogen gas bubbling from the negative electrode and oxygen being given off by the positive electrode. These are products of the electrolysis of water. This is the first sign that current is flowing through electrophoresis system. Shortly afterwards bands of loading dye should be seen moving into the gel and migrating towards the positive pole of the apparatus. ...