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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}