DNA Isolation

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assays/DNA Isolation/protocols/DNAIsolation.md

+## DNA isolation
+
+For DNA extractions, root and soil samples were homogenized in a Precellys 24 TissueLyser (Bertin Technologies) for 2 x 30 s at 6,200 rpm with 15 s intervals. DNA was extracted with a modified, high-throughput version of the FastDNA SPIN kit for Soil (MP Biomedicals). In brief, samples were taken up in sodium phosphate buffer (MP Biomedicals) and MT buffer (MP Biomedicals), then homogenized as described before.
+After centrifugation for 15 min at 13,000 rpm, 150 μl of the supernatant were transferred to a 96-well Acroprep Advance filter plate (with 0.2 μm Supor filter; Pall). Once full, the filter plate was positioned on a PCR plate filled with 50 μl Binding Matrix (MP Biomedicals) per well and centrifuged for 15 min to remove residual soil particles. This and all subsequent centrifugation steps were carried out in a swing out centrifuge at 1,500 rpm. The PCR plate was sealed and shaken for 3 min to allow binding of the DNA to the Binding Matrix. The suspension was pipetted onto a second filter plate of the same kind, positioned on a collection plate, and centrifuged for 15 min. The flowthrough was discarded. Then, 200 μl SEWS-M washing buffer (MP Biomedicals) were pipetted into each well of the filter plate and centrifuged for 5 min. This washing step was carried out a second time. The flowthrough was discarded and followed by centrifugation for 5 min to  remove residual SEWS-M buffer. Finally, 30 μl nuclease-free water were added to each well and left to incubate at room temperature for 3 min. Subsequent centrifugation for 5 min eluted the DNA into a clean PCR plate. The resulting DNA was used for v5v7 16S rRNA region amplification without prior adjustment of DNA concentrations.
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