Dneasy powerplant pro htp 96 kit

The root DNA samples used in this study were derived from our previous two-year-long monthly HLB survey that was conducted in a ca. 2-hectare block of 4–5 years old grapefruit trees on sour orange rootstock in a commercial orchard in Texas (US plant hardiness zone 10a) [32 (link)]. Fibrous root samples were collected from two locations of each tree, about two feet from the tree trunk, by digging one to five inches deep into the soil (sandy clay loam) [32 (link)]. Prior to root DNA extraction, the collected root samples were air dried for 24 h at room temperature from which excess big soil particles were removed from the fibrous roots by gentle tapping with fingertips. The root samples were chopped to 1–2 mm in length to aid maceration followed by root DNA extraction following the manual of the DNeasy PowerPlant Pro HTP96 kit (Qiagen, Hilden, Germany) [32 (link)]. The prepared root DNA fractions were kept in a −20 °C freezer.

After thawing, rhizosphere and spermosphere washes were concentrated by centrifugation at 15,000 g for 15 min, generating a pellet. The supernatant was removed, and the process repeated until 3 ml of sample had been processed. The pellet was re-suspended in an additional 1 ml of spermosphere or rhizosphere wash, before proceeding with DNA extraction. In contrast, after thawing, 50-ml conical tubes containing roots, shoots, or seeds received five 6.35-mm carbon steel ball bearings and 1 ml of sterile distilled water and were then vigorously shaken by hand until the supernatant obtained the consistency of a thick soup.
Then, 400 μl of these liquid samples was transferred to a 2-ml Eppendorf tube containing five 2.3-mm zirconia/silica beads (Cat#11079125z, Biospec Products, United States) along with 500 μl of Qiagen Powerbead solution, RNAse A, Phenolics Blocker, and Solution SL (Qiagen, United States). These were shaken for 20 min in a Harbil 5G-HD 5 Gallon Shaker (Part#32940, Fluid Management, United States) and then centrifuged at 13,000 RCF for 2 min before up to 700 μl was aspirated off with a pipette and added to buffer IL. The rest of the protocol was followed as per Qiagen instructions with the DNeasy PowerPlant Pro HTP 96 Kit (Qiagen, United States).

Frozen liquid samples (spermospheres and rhizospheres) were centrifuged at 15,000× g for 5 min to concentrate microbial cells as a pellet. Supernatant was taken off and the procedure repeated until 3 mL of sample had been concentrated. The resulting microbial pellet was resuspended in an additional 1 mL of unfrozen rhizosphere or spermosphere. On the other hand, after unfreezing, 1 mL of sterile, distilled water and five 6.35 mm carbon steel ball bearings were added to shoots, roots and seeds in tubes, followed by hand-shaking until the liquid took on the consistency of thick soup.
A total of 400 uL was taken from these slurries and transferred to 2 mL microcentrifuge tubes containing five 2.3 mm zirconia/silica beads (Cat#11079125z, Biospec Products, Bartlesville, OK, USA) and RNAse A, Phenolics Blocker, and Solution SL 500 uL of Qiagen Powerbead solution (Qiagen, Germantown, MD, USA). For 20 min, these samples were shaken using a Harbil 5G-HD 5 Gallon Shaker (Part#32940, Fluid Management, Wheeling, IL, USA), followed by centrifugation for 2 min at 13,000 RCF. A total of 700 µL of the supernatant was transferred to a fresh tube. The rest of the protocol was followed as per Qiagen instructions with the DNeasy PowerPlant Pro HTP 96 Kit (Qiagen, USA).