Magattract magnetic beads

After 8 days since the transfer of germinated seeds into Hoagland solutions, fresh leaves were collected from each pot and processed to extract DNA. For each leaf sample, 50 mg was homogenized with 300 µL of RLT buffer in two ml Eppendorf tubes using a Tissue Lyser (Qiagen, Hilden, Germany) for 5 min at 30 hertz. The tubes were centrifuged at 6000 rpm for 3 min, and the supernatant obtained was processed using Biosprint 96 (Qiagen, Hilden, Germany) to extract DNA. The automatic extraction involved the use of six 96 well S-Block plates set up as follows. One plate filled with 300 µL of sample supernatant, 200 µL of isopropanol, and 20 µL of MagAttract magnetic beads (Qiagen, Hilden, Germany). A second plate with 500 µL of buffer RLT. The third and fourth plates with 500 µL of 96% ethanol. The fifth plate with 500 µL of 0.02% Tween solution and the last plate filled with 100 µL of nuclease-free water. After the extraction process, DNA was quantified with a Qubit fluorimeter (Thermo Fisher Scientific Inc., Waltham, MA, USA) using the Qubit™ DNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

DNA was extracted from 50 mg of fresh leaf material. Samples were homogenized inside the collection microtubes with 300 μl of Buffer RLT and 3 mm stainless steel beads. The homogenization step involved the use of Tissue Lyser (Qiagen, Hilden) for 5 min at 30 Hz. Homogenized samples were then transferred in a 96-well S-block plate containing also 200 μl of isopropanol and 20 μl of MagAttract magnetic beads (Qiagen). This plate was used for automatic DNA extraction using Biosprint 96 (Qiagen) together with five other plates respectively composed of 500 μl of Buffer RPW, 500 μl of 0.02% Tween, and two plates filled with 500 μl of 96% ethanol. DNA was eluted in 100 μl of nuclease-free water. Nucleic acid quantification was performed using Qubit (Thermo Fisher Scientific, Carlsbad, CA) with Qubit DNA High Sensitivity Assay Kit (Thermo Fisher Scientific).