title: qiime_experiment_1754:684387:84
design: temporal variablity of soil bacteria across land-uses
library_construction_protocol: DNA was extracted with a portion of the 16S rRNA gene amplified and pyrosequenced according to methods described previously (Lauber et al., 2009). Briefly, 0.1-0.2 g of soil was loaded into bead tubes containing solution C1 of the MoBio Power Soil DNA extraction kit (MoBio Laboratories, Inc., Carlsbad, CA, USA), incubated for 10 min at 65?C and then vortexed for 2 min at maximum speed with the MoBio vortex adapter. The remaining steps of the extraction procedure were performed as directed by the manufacturer. The V4-V5 regions of the 16S rRNA gene were amplified from the extracted DNA using primers designed to amplify bacteria and archaea with few biases against individual taxa (Bates et al., 2011; Bergmann et al., 2011). The primers anneal to positions 515 and 806 (based on E. coli 16S rRNA numbering) and contain the A and B 454 FLX adapters and a 2-bp linker 5? to the 16S-specific portion of the oligonucleotide. In addition, the forward primer contains a unique 12 bp barcode to allow sequences to be assigned to specific samples. All PCR reactions were performed in triplicate using 1X 5 Prime Hot Master Mix (5 PRIME Inc., Gaithersburg, MD, USA), 5 pmol forward and reverse primers, and 2 ml template DNA (Bates et al., 2011). Amplicons from replicate PCR reactions were pooled and cleaned using the MoBio PCR clean up kit and quantified using a Picogreen dsDNA assay (Life Technologies, Grand Island, NY, USA). An equal amount of amplified DNA from each sample was combined into a single tube and sequenced at Engencore (University of South Carolina) on a Roche 454 GS-FLX-Titanium sequencer.
identifiers.namespace: CCME-COLORADO
identifiers.id: qiime_experiment_1754:684387:84