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Introduction of Gel Electrophoresis

What is a plasmid?

Gel Electrophoresis, A plasmid is a circular double stranded structure of DNA molecule which is separate from a cell’s own chromosomal DNA. Plasmids are found in bacteria, yeast and some eukaryotic organisms. Plasmids range from a few thousand base pairs to 100 kilobases (kb). When an organism containing plasmid divides, the plasmid is also replicated before every cellular division. Plasmids exist in a symbiotic relationship with their host. They provide some benefit to the host especially resistance from antibiotics. Drug resistance plasmids make their host resistance to the mechanisms of several drugs. Plasmids also contain transfer genes which code for certain proteins and can be transferred from one organism of the same species to another. This transfer results in wide spread resistance of the bacteria or other species to a drug in a certain setting such as a hospital (Wilson and Hunt, 2002).

Spectrophotometric analysis of DNA

The amount of nucleic acid in a sample can be checked by the use of UV spectrophotometry. DNA and RNA both absorb UV light effectively which makes it possible for the analysis to detect the presence of nucleic acid even if the concentration is extremely low. Spectrophotometric analysis uses the principle that nucleic acids absorb UV light in a specific pattern and if present, the emergent rays striking the photo detector will be less than the incidence rays and hence produce a higher optical density. The value of the optical density obtained will depend on the concentration of the DNA or RNA present in the sample (Huss, Festl and Schleifer, 1983).

Gel electrophoresis, how can the plasmid DNA be visualized on an agarose gel?

Gel electrophoresis is a technique which is used to separate DNA according to its size for the purpose of visualization and purification. The procedure requires an electric field which moves the negatively charged nucleic acid towards the positively charged electrode. The fragments of DNA move towards the electrode at the speed depending on their molecular weight. Hence the shorter DNA fragments will move faster along the gel matrix than the ones longer in size. Therefore the exact length of each fragment can be determined by running them alongside a DNA ladder which contains DNA fragments of known lengths. In order to visual the DNA, ethidium bromide is added. When it binds to the DNA fragments it allows the user to visualise the fragments under UV light (Addgene, 2015).

What is a restriction enzyme?

In order to clone a specific part of the DNA present in a plasmid molecule, the fragments must be extracted out and then inserted into vector DNA. Restriction enzymes are present in the bacteria which recognise certain sequences of nucleic acid which are known as restriction sites. The enzymes then cleave the DNA at those specific sites. These enzymes cleave the nucleic acid from within the DNA molecule…