JoVE-Enzyklopädie der Experimente
Mikrobiologie
0 Aufrufe • 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 millimolar calcium chloride, and optionally, with 25 micrograms per milliliter of kanamycin, to an optical density at 600 nanometers of about 1.0. Make a dilution series of an existing P1 phage stock in the LB medium, and mix 200 microliters of the bacterial suspension with 100 microliters each phage dilution in individual 15 milliliter centrifuge tubes. Incubate the tubes statically for 20 minutes at 37 degrees Celsius.
At the end of the incubation, add approximately three milliliters of molten top agar, supplemented with ten millimolar calcium chloride to the tubes. Mix the contents by vortexing, and pour the mixtures onto pre-warmed LB plates to make even layers. Then, incubate the plates overnight at 37 degrees Celsius.
The next morning, select a plate with a semi-confluent growth of phage plaques and use an inoculation loop to scrape the top agar layer into a centrifuge tube. Under fume hood, add one to two milliliters of LB and a drop of chloroform to the tube, and vortex the tube vigorously for one minute. Pellet the agar and bacterial cells by centrifugation and transfer the supernatant to a microcentrifuge tube, avoiding debris from the pellet.
Then, add two drops of chloroform and store the lysate at four to ten degrees Celsius.