Laboratory 3, 4, 5 Milomir Janjic
Purification, Quantitation, Structure and Modification of Plasmid DNA
October 22, 2012
Plasmids are found in a many bacterial species. Bacterial plasmids are closed circular extrachromosomal molecules of double-stranded DNA. They behave as separate genetic units and are replicated independently of the chromosomal DNA. They are much smaller than chromosomal DNA in size and range from 1 to 200kb. Plasmid DNA has been recognized as the most powerful tool in molecular cloning because of their simplicity and practicality in propagating foreign genes. In research, plasmid DNA is used as a vector allowing the study and generation of Genetically Modified Organisms (GMO).
The bacterial strain used as host in our experiment was GMO endA negative E coli strain. The wild type strain usually contains endA gene, which is responsible for production of endonucleasa I. This protein is part of bacterial antiviral defense mechanism and it can destroy double-stranded DNA. Therefore, to prevent lower yield of transfection, we used as host GMO endA negative E coli strain.
There are several ways to purify plasmids. Plasmid DNA isolation techniques can be simple - low quality DNA preparations “minipreps” and more complex, time-consuming high quality DNA preparations. The high quality DNA preparation requires organic or hazardous cesium chloride ingredients, and the entire process can take a few days. As a result, we used much safer “minipreps” method to isolate DNA in less than three hours. Minipreps involve lysing the cells, purifying the DNA via centrifugation, and finally, concentrating the plasmid particles.
The quantitative DNA analysis was also performed in this experiment. To measure amount of DNA in our purified, dilute sample (i.e., without significant amounts of contaminants such...