Removal and DNA Extraction of Phyllosphere Microbes
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- This protocol based directly on A Direct Method to Isolate DNA from Phyllosphere Microbial Communities Without Disrupting Leaf Tissues. W Suda, M Oto, S Amachi, H Shinoyama, M Shishido. Microbes Environ. Vol. 23, No. 3, pg. 248-252, 2008.  Minor changes were made to the tenses and placements of verbs for protocol clarity.
- Place 5 g non-shredded fresh leaf sample in a 50 mL sterilized polypropylene tube. Add 5 mL of extraction buffer (100 mM Tris-HCl, pH 9.0, 40 mM EDTA), 1 mL of 10% SDS, and 3 mL of benzyl chloride.
- Incubate the tube for 15 min at 50°C by mixing repeatedly with 1-min intervals so that the two phases are thoroughly mixed.
- Remove leaves from the tube, and add 3 mL of 3M sodium acetate (pH5.0).
- Incubate on ice for 10 min, then centrifuge the suspension (6000xg, 15 min, 4°C).
- Transfer the aqueous phase to a new centrifuge tube, and precipitate DNA by adding an equal volume of isopropanol followed by centrifugation (9000xg, 15 min, 4°C).
- Air-dry pellet, then resuspend in 200 uL of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0).
- To purify crude DNA extract, use gel filtration. A protocol for gel filtration can be found here ( https://www.nature.com/articles/nmeth0506-411 )
Removal of Live Phyllosphere Microbes for Later Inoculation
- Attach "nubby" vortex head to the vortexer. Prepare at least 5 TSA plates for later use.
- Create a 0.01% solution of tween 80 in sterile PBS. As of now, use a working ratio of 5 mL washing solution to 1 g of plant matter.
- A very simple way to create this solution is to add 1 uL of Tween80 to each 10 mL of PBS! For example, add 4 uL of Tween80 to 40 mL of sterile PBS. Vortex thoroughly!
- Using sterile scissors and sterile forceps (sterilized with ethanol OR flame, whichever is the easiest and most effective), hold the individual Arabidopsis by a leaf and snip the stem just above the surface of the soil - transfer to sterile 15 mL conical tube labelled with the sample(s)’ s ID and date obtained.
- Add 0.01% tween 80 solution to conical tube containing plant matter - use the ratio’s given above! If necessary, close the cap on the conical tube and weigh the tube with plant matter compared to an empty tube, then make calculations.
- Tape the tube to the nubby vortex head - set the vortexer to around 7 or 8 speed and allow to be shaken for 2 minutes.
- After vortexing 2 minutes, take 10 uL of original shaken solution and pipet onto pre-marked surface of the TSA plate.
- Also remove 10 uL of the original solution and dilute 1:10 in sterile water three times to create 10^-1, 10^-2, and 10^-3 aliquots - also pipet 10 uL of each aliquot onto the pre-marked TSA plate surface.
- Repeat the above steps for the same conical tube for a total of 4, 6, and 8 minutes of shaking.
- Repeat the above steps with a fresh sample and a fresh conical tube, each time using different TSA plates for each iteration of the experiment.
- Allow plates to grow at or above room temperature for 2 to 3 days, monitoring at least once a day and recording observations.