JoVE Encyclopedia of Experiments
Microbiology
0 views • 5:12 min • March 31st, 2026
Begin with small pieces of chilled leaf tissue in which the phloem is infected with a bacterial pathogen.
Place the pieces in a mortar with liquid nitrogen to inhibit the activity of DNA-degrading nucleases.
Grind to mechanically disrupt the cells and release intracellular components.
Use a chilled spatula to transfer the powder, add lysis buffer, and vortex the suspension.
Incubate to lyse plant nuclei and bacterial cells, releasing DNA and RNA while denaturing proteins.
Add RNase and incubate to degrade RNA.
Add protein precipitation buffer to aggregate proteins, then centrifuge to separate them.
Transfer the supernatant containing plant and bacterial DNA to isopropanol to precipitate the DNA as thread-like strands.
Centrifuge to collect DNA and discard the supernatant.
Wash with ethanol, then centrifuge, and discard the supernatant.
Air-dry the DNA, then add a rehydration buffer and incubate to dissolve it.
The DNA samples are now ready for downstream PCR-based pathogen detection.
Use clean scissors to cut a whole fresh leaf from a citrus tree, and place in a clean plastic sandwich bag. Then, chill a mortar and pestle by adding small quantity of liquid nitrogen. Next, use the scissors to cu
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