JoVE Encyclopedia of Experiments
Microbiology
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Begin with a transfection mix containing a fine precipitate of plasmids with calcium and phosphate ions.
These plasmids encode viral proteins and the gene of interest.
Add this solution dropwise onto a monolayer of embryonic kidney cells.
Gently swirl the plate to ensure uniform distribution.
Incubate the cells to allow binding of the precipitate to the cell surface.
Now add serum to the medium to support cell survival.
Calcium and phosphate ions help internalization of the plasmid-precipitate via endocytosis.
The plasmids then enter the nucleus, and the plasmid-encoded genes are expressed to produce viral proteins.
The plasmids are also transcribed to generate the viral RNA genome containing the target gene.
These components assemble into viral particles within the cytoplasm.
Later, recombinant viral particles carrying the gene of interest bud from the plasma membrane and release into the culture medium, enabling continued production over time.
Add 2.5 milliliters of plasmid mixture in drops to each of the 15-centimeter plates. Then, gently swirl the plates and incubate at 37 degrees Celsius with 5 % carbon dioxide for two to three hours.
After three hours, add 2.5 milliliters of 10 % serum to each plate and continue for overnight incubation.