Genetics Lab

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Genetics Lab


Introduction:
The objective of this lab is to prepare a genomic library that will be later screened to obtain a clone that contains (LUX) operon of the studied bacterium “vibrio fischeri”. This requires several steps including DNA isolation, restriction digestion, electrophoresis and then ligation. The results from the experiments above can be used in many interesting ways, not only to clone known genes, but also to disclose the limitless amount of undiscovered genes that have potential in medicine and gene therapy.
Also, it presents a system that allows performing the isolation of the genomic library of bacterium “vibrio fischeri” clone and preparation and study of the plasmid subclone in a short period of time.
Isolation of plasmid DNA involves: growth of bacteria and amplification of plasmid, harvesting and lysis of bacteria, and purification of plasmid DNA.
DNA isolation from a particular organism requires celll lysis. Lysozyme enzyme is used to allow access to peptidoglycan and degrade it so that isolation of DNA can be facilitated. The most important reagents used in DNA isolation are: phenol and chloroform that dissociate protein from nucleic acid. Ethanol is very important to precipitate DNA provided and ionic strength of the solution is high and DNases are inactivated. Then, the resulting DNA is highly purified.
Spectrophotometric analysis of DNA is used to quantify DNA. i.e: finding the concentration of the isolated DNA. This concentration depends on the purity of DNA isolated. Spectrophotometric measurements were taken at different wavelengths to compare ratios with the ideal ones. Hence, contamination with RNA molecules or proteins is to be used to initiate the cloning of (LUX) genes.
Restriction digestion is used in making genomic libraries. In addition, identification of differences in restriction enzyme. Cleavage patterns between individuals has become an important tool for the diagnosis ...