Isolation of Badnavirus Particles from Infected Leaves

Take a frozen leaf infected with Badnavirus and cut it into small pieces.

Add a grinding buffer and sodium sulfite. Grind the pieces at a low temperature.

The grinding buffer maintains stable extraction conditions, while sodium sulfite prevents oxidative damage.

Transfer the homogenate, and add urea and a nonionic detergent.

Incubate with stirring at a low temperature to maintain virus stability.

Urea degrades cell components, and the detergent destabilizes membranes, releasing the virions.

Transfer the mixture and centrifuge to pellet debris.

Next, filter the supernatant. Transfer and centrifuge again to form two pellets: a white virion pellet and a green plant debris pellet.

Discard the supernatant. Scrape the white pellet and resuspend it in water. Place it on ice and incubate for complete solubilization.

Centrifuge at low speed to remove residual debris.

Collect the supernatant and centrifuge at a higher speed to pellet the virions for further analysis.

A laboratory coat and gloves should be worn for all steps of this procedure. Start by cutting 80 to 100 grams of leaves from diseased plants previously frozen at minus 20 degrees Celsius. Then grind the leaves in a Waring blender using 200