From c8f268788fdbc3ed16437095beb365dcff3ce95a Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Thu, 20 Mar 2025 14:53:08 +0100 Subject: [PATCH] DNA Isolation --- .gitattributes | 1 + assays/DNA Isolation/README.md | 0 assays/DNA Isolation/dataset/.gitkeep | 0 assays/DNA Isolation/isa.assay.xlsx | Bin 0 -> 9419 bytes assays/DNA Isolation/protocols/.gitkeep | 0 .../DNA Isolation/protocols/DNAIsolation.md | 4 ++++ .../isa.assay.xlsx | Bin 13480 -> 10656 bytes 7 files changed, 5 insertions(+) create mode 100644 assays/DNA Isolation/README.md create mode 100644 assays/DNA Isolation/dataset/.gitkeep create mode 100644 assays/DNA Isolation/isa.assay.xlsx create mode 100644 assays/DNA Isolation/protocols/.gitkeep create mode 100644 assays/DNA Isolation/protocols/DNAIsolation.md diff --git a/.gitattributes b/.gitattributes index e09f562..eac0854 100644 --- a/.gitattributes +++ b/.gitattributes @@ -13,3 +13,4 @@ assays/InvitroIronMobilizationAssay/dataset/.gitkeep 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diff --git a/assays/DNA Isolation/protocols/.gitkeep b/assays/DNA Isolation/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/DNA Isolation/protocols/DNAIsolation.md b/assays/DNA Isolation/protocols/DNAIsolation.md new file mode 100644 index 0000000..abb6f07 --- /dev/null +++ b/assays/DNA Isolation/protocols/DNAIsolation.md @@ -0,0 +1,4 @@ +## 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. 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