Supplementary Information 1
Site description, soil collection and soil treatments
Landfill soil was collected from a local landfill site in Ufton, UK (latitude 52° 15' 0
N; longitude 1° 25' 60 W) in April 2008. The vegetation, predominantly grass above
the cover soil, was cleared before collecting soil to a depth of 30 cm. The moisture
content of the soil at the time of sampling was measured by oven drying soil subsamples (after removing stones) at 80oC until constant weight (26.4% ± 1.3). The soil
was stored at 40C until use.
Nucleic acid extraction, purification and cDNA synthesis
Total nucleic acids were extracted using 0.5 g of soil sub-samples from time 0, I and
II by employing a modified bead beating protocol described by Chen et al. (2007).
Nucleic acid extractions for each soil treatments were done in triplicate (single
extraction from two 13C- and one 12C-CH4 soil incubations in serum bottles). RNA
samples were confirmed to be DNA-free by lack of amplification of 16S rRNA genes
with the universal bacterial primer set 27f/907r (Lane, 1991; Muyzer et al., 1993) (30
cycles of 94oC for 1 min, 60 oC for 1 min and 72 oC for 1 min with a final extension at
72 oC for 10min). Nucleic acid concentrations were quantified using a Nanodrop
spectrophotometer (Nanodrop). SuperScriptTM II Reverse Transcriptase kit
(Invitrogen) was used to synthesize cDNA from 100 ng of RNA, following the
manufacturer’s instructions, using primers mb661r for pmoA (Costello and Lidstrom,
1999). cDNA generated was used as a template for PCR (35 cycles) using pmoA
primers A189f/ mb661r (Costello and Lidstrom, 1999; Holmes et al., 1999) at an
annealing temperature of 55oC to confirm the reverse transcription reaction. Control
reactions were prepared as described above, but without reverse transcriptase to check
for DNA contamination in RNA samples. Replicate RNA samples (two 13C-CH4 soil
incubations in serum bottles) from each soil treatments were used to synthesize cDNA
representing pmoA genes. These samples were subsequently pooled and subjected to
pmoA microarray analysis.
DNA-Stable isotope probing
DNA extracted from time II was used for DNA-SIP analysis. One microgram of DNA
extracted at time II (pooled DNA from two 13C-CH4 soil incubations) was used for
density gradient ultra-centrifugation followed by gradient fractionation, precipitation
of DNA and buoyant density measurements of the fractions as described by Neufeld
et al. (2007).
pmoA microarray analysis
PCR amplification of pmoA genes from DNA and cDNA were performed with primer
set A189f/T7-mb661r (Bourne et al., 2001) in duplicate reactions (from two 13C-CH4
serum bottles), which were subsequently pooled following PCR. PCR was carried out
over 35 cycles at 95oC for 1 min, 55oC for 1 min, 72oC for 1 min, and a final
extension step at 72oC for 10 min in a Biorad thermal cycler. Each PCR reaction
contained 1× reaction buffer, 0.2 mM dNTPs, 2.5 mM MgCl2, 0.2 M of each primer
(forward and reverse) (Invitrogen) and 2.5 units of Taq DNA polymerase
(Fermentas). In vitro transcription, RNA purification and microarray hybridisation for
DNA were performed according to Stralis-Pavese et al (2004) and for mRNA as
described by Bodrossy et al (2006). After hybridization, slides were scanned using a
GenePix 4000 A laser scanner (Axon) at 10 mm resolution at 532 nm wavelength
(Cy3). Results were then normalized to a positive control probe (mtrof 173)
(Bodrossy et al., 2003).
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