Ashley Campbell edited Methods.tex  over 10 years ago

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\textbf{Nucleic Acid Processing}  DNA was extracted from 0.25g soil using a modified Griffiths procotol (Griffiths 2000 Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition, 10.1128/AEM.66.12.5488-5491.2000). Soils were bead beat in 2mL lysis tubes containing 0.5g silica/zirconia beads (treated at 300°C for 4 hours to remove RNases), 0.5mL extraction buffer (240 mM Phosphate buffer  0.5\% N-lauryl sarcosine), and 0.5mL phenol-chloroform-isoamyl alcohol (25:24:1) for 1 min at 5.5 m s ^{-1}. After beat beading, 85$\mu$L 5M NaCl and 60$\mu$L 10\% hexadecyltriammonium bromide (CTAB)/0.7M NaCl were added to lysis tube, vortexed, chilled for 1min on ice, and centrifuged at 16,000 x g for 5min at 4°C. The aqueous layer was transferred to a new tube and reserved on ice. To increase DNA recovery, a double extraction was performed by adding 85$\mu$L 5M NaCl and 0.5mL extraction buffer to same lysis tube, vortexed, and centrifuged same as before. New aqueous layer was added to previously collected aqueous layer and washed with 0.5mL chloroform:isoamyl alcohol (24:1), vortexed, and centrifuged same as before. Aqueous layer was transferred to a new tube and nucleic acids were precipitated using 2 volumes polyethylene glycol solution (30\% PEG 8000, 1.6M NaCl) on ice for 2hrs, followed by centrifugation at 16,000 x g, 4°C for 30min. Supernatant was discarded and pellets were washed with 1mL ice cold 70\% EtOH; each wash being a vortex followed by centrifugation at 16,000 x g, 4°C for 10min. Pellets were air dried, resuspended in 50uL 50$\mu$L  TE, and replicate extractions pooled. Nucleic acids were stored at -20°C. To prepare for DNA-stable isotope probing (DNA-SIP), DNA was size selected (>4kb) using 1\% low melt agarose gel and $\beta$- agarase I enzyme extraction per manufacturers protocol (New England Biolab, M0392S)