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. ...
The movement of the DNA fragments are highlighted by the dye which forms coloured bands.(See above and below)
Results: Depending on the endonuclease used, the digested DNA samples, exhibit the characteristic bands produced by each restriction. The enzyme is seen because of a dye that is introduced and shows the pattern of movement of the DNA fragments.
Various restriction enzymes: There are several types of restriction enzymes, each of which react differently with the DNA and create different cuts. there are some that cut a three base pair sequence while others can cut four six or even eight. Each enzyme has different properties that are factors that determine the efficiency with which the DNA is cut and under what conditions optimum results will be obtained. Manufacturers of these restriction enzymes usually provide a specific buffer solution that is unique to the enzyme. This buffer solution is a mix of cat ions and other components that optimize the efficiency of the enzyme for cutting. Different restriction enzymes have different optimal temperatures under which they function.
Conclusion: It can be observed that each sample of purified DNA reacts differently with the varied restriction enzymes. The fourth sample used for the negative control remains intact; as it has been incubated without, an endonuclease.The new DNA fragment can then be extracted from the gel by cutting it and doing gel purification. Vector ligation is used as the insert point. The ligation of the vector has to be cut also by a restriction