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 tissue, approximately one square inch in size.

Place the leaf pieces in the mortar to freeze them immediately. Quickly start grinding the leaf tissue with the mortar until the liquid nitrogen evaporates, and a fine green powder remains. Next, chill a metal spatula in liquid nitrogen for 10 to 15 seconds. Then use the cold spatula to scoop the ground tissue from the mortar in a 1.5 milliliter micro-centrifuge tube.

Use a 1000 microliter pipette to add 600 microliters of nuclei lysis solution to the sample. Then, vortex the sample for one to three seconds. Incubate the tube at 65 degrees Celsius, in a water bath for 15 minutes.

Once the incubation period is over, use a 10 microliter pipette to add 3 microliters of RNase solution to the lysate to remove RNA. Then, mix the contents of the tube by inverting it two to five times.

Incubate at 37 degrees Celsius for 15 minutes in a cabinet incubator. After 15 minutes, add 200 microliters of protein precipitation solution to the sample. Vortex the tube at high speed for 20 seconds. Next, centrifuge the sample at 13000 g for three minutes.

During the course of centrifugation, add 600 microliters of isopropanol maintained at room temperature to a new 1.5 milliliter microcentrifuge tube. After the centrifugation is over, carefully remove the supernatant from the centrifuge tube, to the tube containing isopropanol. Discard the pellet.

Invert the tube for the DNA to be visible, like a thread-like strand. Next, centrifuge the sample at 13000 g for a minute at room temperature.

Decant the supernatant after centrifugation. Then, add 600 microliters of 70% ethanol to the pellet. Invert the tube several times to wash the pellet.

Centrifuge the tube at 13000 g for one minute. After the centrifugation is over, carefully aspirate the supernatant. Then invert the tube with the lid open on an absorbent paper to air dry for 15 minutes.

Once dried, add 100 microliters of DNA rehydration solution to the pellet. Incubate the DNA suspension at 65 degrees Celsius in a water bath for an hour. Tap the tube periodically while mixing.

Add 1 microliter of purified water in a micro-cuvette. Read the water as blank on the spectrophotometer. Then, add 1 microliter of the sample in the microcuvette. Place the microcuvette on the spectrophotometer, and obtain the DNA concentration.