Removal and DNA Extraction of Phyllosphere Microbes

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. [1] Minor changes were made to the tenses and placements of verbs for protocol clarity.

1. 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.
2. Incubate the tube for 15 min at 50°C by mixing repeatedly with 1-min intervals so that the two phases are thoroughly mixed.
3. Remove leaves from the tube, and add 3 mL of 3M sodium acetate (pH5.0).
4. Incubate on ice for 10 min, then centrifuge the suspension (6000xg, 15 min, 4°C).
5. 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).
6. Air-dry pellet, then resuspend in 200 uL of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0).