JoVE Enciclopedia degli esperimenti
Microbiologia
0 visualizzazioni • 3:36 min. • July 31st, 2026
Begin with a bacterial strain carrying a selectable marker in a medium supplemented with calcium ions.
Add bacteriophages.
Incubate briefly to allow calcium-dependent phage adsorption and DNA injection.
Add molten soft agar supplemented with calcium ions, vortex the mixture, overlay it onto pre-warmed agar plates, and incubate.
Phage-encoded nucleases degrade the host DNA, while the phage genome replicates, produces proteins, and assembles new infectious particles.
Some phages accidentally package host DNA, including the selectable marker, forming transducing particles.
The host cell lyses and releases both infectious and transducing particles.
Infectious particles spread to neighboring cells, creating plaques.
After incubation, scrape the soft agar layer containing the phages and transfer it to a tube.
Add a medium with chloroform and vortex thoroughly to lyse the residual host cells.
Centrifuge to remove debris and collect the supernatant.
The resulting phage lysate contains transducing particles for further analysis.
To infect a donor strain containing a gene deletion based on an excisable antibiotic cassette, first, grow the bacteria in 5 milliliters of lysogeny broth, or LB, with ten mill
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