Introduction to Recombinant Genetics- Biology 350
Chapter Review - Phage isolation
Background on bacteriophages
Bacteriophages can serve as a source of cloned DNA.
Two types of bacteriophages are commonly used, the lambda phage and the M13 family of phages.
The lambda phage can exist as a prophage (integrated into bacterial genome, lysogenic) or as an independent genetic element (lytic). The lambda phage is a linear double-stranded bacteriophage when packaged in the phage protein coat. The lambda DNA has 13 unpaired complementary bases at each end of the linear DNA and upon infection of a cell it circularizes.
The M13 phage enters the bacteria through pili and mature phage buds from the cell without lysing the bacteria. M13 is a single-stranded DNA phage which produces a double-stranded replicative form. The double-stranded replicatitive form of the M13 phage can be isolated from the bacteria by standard plasmid isolation proceedures.
Many Phages can be detected by plating them on a bacterial lawn and looking for cleared regions (plaques) where the bacteria have been lysed (clear plaques, lambda) or the bacterial grown has been slowed (cloudy plaques, M13).
Isolation of DNA from bacteriophages
Isolation of DNA from bacteriophages begins by separating the phage from the cellular components. Since active production of phage results in the release of phage particles into the media, phage particles can be separated from cells by centrifugation. Phages are small and will be retained in the supernatant while cells and cellular debris will be pelleted.
Phages can be isolated from the supernatant with the addition of PEG (polyethylene glycol) and NaCl. The PEG absorbs water and causes the phage to aggregate and precipitate. After centrifugation the precipitated phage is in the pellet.
Phage can be further purified in a CsCl gradient (NO Ethidium Bromide). The phage can easily be visualized in the gradient by shining a flashlight on the gradient in a darkened room and looking for the light scatter created by the phage particles.
Since both lambda and the M13 phages are isolated with their protein coat, you must remove the protein coat in order to purify the DNA.
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