Extraction of Total DNA from Leaves for Detection of Bacterial Infection

Samenvatting

Source: Chen, H., et al. Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples. J. Vis. Exp. (2018)

This video demonstrates the extraction of total genomic DNA from citrus leaf tissue infected with a pathogenic bacterium. The protocol enables reliable DNA recovery for downstream PCR-based detection of the phloem-limited pathogen.

Protocol

1. Isolate Genomic DNA from Plant Tissue Using a Genomic Extraction Kit

1. Obtain citrus leaf tissue by cutting a whole fresh leaf from a citrus tree using clean scissors, and place the leaf in a clean plastic sandwich bag.
   NOTE: The bag containing the leaf tissue should be placed in a 4 °C refrigerator or on ice in an insulated container as soon as possible after collection to avoid spoilage.
2. Add a small amount of liquid nitrogen to chill a mortar and pestle. While they are chilling, use scissors to cut out a small piece of leaf tissue, approximately 1 square inch in size. Place the cut tissue in the mortar to instantly freeze it. Add more liquid nitrogen, if needed.
   NOTE: Insulated gloves should always be worn while handling liquid nitrogen.
3. Quickly grind the plant tissue into a fine powder using the mortar. Continue grinding until the liquid nitrogen completely evaporates, and a fine green powder remains.
4. Briefly chill a metal spatula in liquid nitrogen for 10 - 15 s, then scoop the ground tissue inside the mortar into a 1.5 mL microcentrifuge tube, using the spatula.
5. Add 600 µL of Nuclei Lysis Solution (see Table of Materials) using a 1000 µL pipette. Vortex the sample for 1 - 3 s, then incubate in a 65 °C water bath for 15 min.
6. Add 3 µL of RNase Solution to the lysate using a 10µL pipette, invert the tube 2 - 5 times to mix it, and incubate at 37 °C in a cabinet incubator for 15 min.
   NOTE: Allow the mixture to cool to room temperature before proceeding.
7. Add 200 µL of Protein Precipitation Solution, vortex at high speed for 20 s, then centrifuge for 3 min at 13,000 x g to form a firm pellet. While the sample is being centrifuged, add 600 µL of room-temperature isopropanol to a fresh 1.5 mL microcentrifuge tube.
8. Using a 1000 µL pipette, carefully remove the supernatant from the centrifuged sample, transferring it to the fresh 1.5 mL microcentrifuge tube containing isopropanol. The pellet may now be discarded.
   NOTE: Avoid contaminating the supernatant with protein by leaving a minuscule amount of supernatant above the pellet.
9. Invert the tube until the DNA becomes visible as a mass of thread-like strands, then centrifuge at 13,000 x g for 1 min at room temperature.
10. Decant the supernatant, add 600 µL of 70% ethanol to the pellet, invert the tube several times to wash the DNA, and centrifuge at 13,000 x g for 1 min.
11. Carefully aspirate the ethanol, leaving the loose DNA pellet, then open and invert the tube, resting on absorbent paper. Air dry at room temperature for 15 min.
12. Add 100 µL of DNA Rehydration Solution to the dried DNA pellet, then incubate the DNA in a 65 °C water bath for 1 h, while mixing periodically by tapping the tube.
13. Place 1 µL of purified water in a microcuvette, and insert it into a spectrophotometer to be used as a blank. Calculate the concentration of DNA present by placing 1 µL of the sample in the microcuvette and inserting it into the spectrophotometer. DNA concentration (ng/µL) can be calculated as (A₂₆₀ - A₃₂₀) × dilution factor × 50 ng/µL.
    NOTE: The protocol can be paused here. The sample can be stored at 4 °C.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Wizard Genomic DNA Purification Kit	Promega	A1120	Source of Nuclei Lysis, RNase, Protein Precipitation, and DNA Rehydration solutions
Liquid nitrogen	Air Products	N/A
5424 table top centrifuge	Eppendorf	05-403-93
Isotemp 215 digital water bath	Fisher scientific	15-462-15Q
Metal spatula	Sigma-Aldrich	Z283274
Mortar and pestle	Sigma-Aldrich	Z247464
LSE Vortex Mixer	Corning	6775
2-propanol	Sigma-Aldrich	I9516
Sterile 10 μL tips	TipOne	1161-3730
Sterile 200 μL tips	TipOne	1163-1730
Sterile 1250 μL tips	TipOne	1161-1750
Variable Volume Pipettor Kit	VWR	75788-460
Tris base	Sigma-Aldrich	10708976001
1.5 mL microcentrifuge tubes	Thermo Fisher Scientific	3439