Extraction of Genomic DNA from Plant Leaf Tissue Infected with Pathogenic Bacteria

Take a leaf infected with Candidatus liberibacter, a pathogenic bacterium.

Cut it into pieces, flash-freeze in liquid nitrogen, and grind to rupture the plant cells and release their contents, including the bacteria.

Add a nuclei lysis buffer and mix thoroughly.

Incubate at a high temperature to lyse the membranes, releasing genomic DNA from plant nuclei and bacterial cells.

Add RNase enzyme, invert the tube to mix, and incubate to degrade RNA.

Add a protein precipitation solution and mix to promote protein aggregation. Centrifuge to pellet protein and cellular debris.

Transfer the supernatant to a tube containing isopropanol and mix to precipitate the DNA into visible strands. Centrifuge to pellet the DNA.

Wash the pellet with ethanol to remove impurities, air dry, and rehydrate the DNA.

Incubate at a high temperature to solubilize the DNA, then load a sample into a microcuvette.

Using a spectrophotometer, measure the isolated DNA concentration.

First, 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 cut small pieces of the leaf ti