Soil nucleic acid extraction with phenol-chloroform (Asteraceae)

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Method synopsis

Modified phenol-chloroform extraction protocol optimized for soil samples using CTAB buffer and mechanical disruption for simultaneous DNA and RNA recovery.

Synthesized from method descriptions across 2 papers using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Virus diversity and activity is driven by snowmelt and host dynamics in a high-altitude watershed soil ecosystem (2023), Microbiome. Implementations in other papers (listed below) may differ.

1. Nucleic acid extraction
   Nucleic acids were extracted on ice from 5 to 7 technical replicates of each soil sample by adding 0.5 mL phenol:chloroform:isoamyl alcohol (25:25:1) to 0.5 g of soil in a 2 ml Lysing Matrix E tube, followed by addition of 0.5 mL of CTAB buffer (5% CTAB, 0.25 M phosphate buffer pH 8.0, 0.3 M NaCl) and 50 μL of 0.1 M aluminum ammonium sulfate.
   Quantities: 5-7 technical replicates per soil sample, 0.5 g soil per replicate, 0.5 mL phenol:chloroform:isoamyl alcohol (25:25:1), 0.5 mL CTAB buffer, 50 μL aluminum ammonium sulfateDuration: Homogenized at 5.5 m/s for 30 s in FastPrep-24 instrument, centrifuged at 16 K g for 5 min at 4°CConditions: Performed on ice, 4°C centrifugation
   Equipment: 2 ml Lysing Matrix E tube, MP Biomedicals FastPrep-24 instrument, MaxTract High Density 2 mL tubes, Qiagen Inc Valencia CA USA
2. DNA and RNA purification
   Samples were extracted a second time as described above, and the aqueous phase from the repeated extractions for each sample was combined. Sodium acetate (3 M sodium acetate, 1/10th volume of total aqueous phase) and ice-cold ethanol (100%, 2X volume of total aqueous phase) were added. DNA and RNA were precipitated overnight at −20 °C, then separated using AllPrep DNA/RNA Mini Kit.
   Quantities: 3 M sodium acetate at 1/10th volume, ice-cold ethanol at 2X volume, overnight precipitation at -20°CDuration: Overnight precipitation, then separation using kit protocolConditions: -20°C precipitation temperature
   AllPrep DNA/RNA Mini Kit (Qiagen Inc., Valencia, CA, USA), Qubit 1X dsDNA Broad Range Kit, Qubit RNA High Sensitivity Kit, 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, USA)

Typical Equipment

• 2 ml Lysing Matrix E tube
• MP Biomedicals FastPrep-24 instrument
• AllPrep DNA/RNA Mini Kit

Output Measurements

• purified DNA
• purified RNA
• nucleic acid concentrations

Papers Using This Protocol (2)

Species Studied With This Protocol

Places Where Used