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 bead beating, 85uL of 5M NaCl and 60uL 10\% hexadecyltriammonium bromide (CTAB)/0.7M NaCl were added to the lysis tube, vortexed, chilled on ice for 1min then centrifuged at 16,000 x g for 5min at 4°C. Aqueous layer was transferred to a new tube and reserved on ice. TO increase DNA recovery, a second extraction was performed by adding 85uL 5M NaCl and 0.5mL extraction buffer to the lysis tube, vortexed, and centrifuged at 16,000 x g for 5min at 4°C. Aqueous layer was added to first aqueous layer collected and washed with 0.5mL chloroform:isoamyl alcohol (24:1) by vigorously vortexing and centrifuging same as before. Aqueous layer was transferred to a new tube where nucleic acids were precipitated using polyethylene glycol solution (30\% PEG 8000, 1.6M NaCl) for 2hrs on ice, followed by centrifugation at 16,000 x g, 4°C for 30min. Supernatant was discarded and pellet was washed twice with 1mL ice cold 70\% EtOH; each wash followed by vortexing then centrifugation at 16,000 x g, 4°C for 10min. After washes, pellet was air dried, resuspended in 50uL TE, then replicate extractions were pooled.     For DNA-stable isotope probing (DNA-SIP), extracted DNA was size selected (greater than 4kb) using 1\% low melt agarose gel and \beta -agarase I per manufacturers protocol (New England Biolab M0392S).  In short, the method involves  bead beating in the presence of cetyl-trimethyl ammonium