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}.In short, the method involves   bead beating in the presence of cetyl-trimethyl ammonium   bromide (CTAB) buffer (1 vol. 10% CTAB in 0.7 M NaCl   mixed with 1 vol. 240 mM phosphate buffer pH 8.0), phenol   and chloroform, followed by phenol extraction and precipita-   tion of nucleic acids from the aqueous phase by 30% poly-   ethylene glycol 6 000 (Fluka, Sigma-Aldrich, Missouri, USA)   in 1.6 M NaCl. The following modifications were applied to the   protocol: (i) Bio101 Lysing Matrix E tubes in combination with   bead beating (FastPrep FP120; MP Biomedicals, California,   USA) were used for cell lysis; (ii) phenol and CTAB buffer   were added to the frozen samples; (iii) cell lysis was per-   formed for 2   ¥   15 s at a speed setting of 5.0 m s   -   1   with inter-   mittent cooling to prevent overheating of the sample; (iv)   samples were kept on ice at all steps in the procedure; (v)   samples were centrifuged for 10 min at 4°C to separate the   aqueous phase containing the nucleic acids from the organic   phase; (vi) following the removal of phenol by chloroform-   isoamyl alcohol (24:1), no more than 400   m   l of the aqueous   phase with nucleic acids was transferred to a new tube to   avoid contamination from the interphase; (vii) after PEG addi-   tion, samples were incubated on ice for 2 h at 4°C; and (viii)   purified precipitated nucleic acids were resuspended in  \textbf{Post-Sequencing Analysis}