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Extraction of Bacteriophage DNA from Large Scale Cultures Using Proteinase K and SDS
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Objectives

 

To extract genomic DNA from Bacteriophage in lysate.

 

Theory

 

Viruses are very small infectious agents and can only be visible by electron microscope. They have an approximate size range of 20 to 300nm ie, 10 to 100 times smaller than most bacteria. Bacteriophage is a virus which infects bacteria by integrating its DNA into the host bacterial chromosome. Different types of bacteriophages are there in the environment each of them can infect a single or limited number of Bacteria. It is difficult to resolve viruses under light microscope. But we can count them using some microbiology culture techniques and the most common method used for counting bacteriophage is Plaque assay. Virus that infects bacteria is called a bacteriophage (literally "bacteria eater"). There are many different kinds of bacteriophages, and each can only infect a single or limited number of bacterial types. Like all viruses bacteriophages have nucleic acids surrounded by a protein coat. The elaborating structure of bacteriophages makes them easy for attaching to the bacterial surface and injecting its nucleic acid to the host cell cytoplasm. Bacteriophages are highly specific in infecting the bacteria. They cause host lysis at the final step of their infection. When the bacterial cells in a broth culture are lysed by a bacteriophage, the turbidity of medium become changes to clear. This is a better indication of host specificity and can be determined by inoculating the bacteriophage into different bacterial culture and then checking for clearing of the media.

 

DNA isolation from bacteriophages begins by separating the phage from its cellular components. This can be  achieved by centrifugation as the active production of phage results in the release of phage particles into the media. Since phages are small they will be retained in the supernatant whereas the cells and cellular debris will be pelleted.

 

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