Extraction of Total DNA from Leaves for Detection of Bacterial Infection

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 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 till 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 3 minutes. During the course of centrifugation, add 600 microliters of isopropanol maintained at room temperature to a new 1.5 milliliter micro-centrifuge 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 1 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.