title: Bacterial Diversity in Seven Terrestrial Sulfidic Springs in the USA
design: DNA extraction was performed using a protocol similar to the commercially available Puregene DNA extraction kit (Gentra Systems, Minneapolis, Minn), with the following modifications: 9 µl of proteinase K (20 mg/ml) was added to each DNA extraction buffer prior to digestion; a freeze-thaw series (three times at -20˚C) was used to aid in disruption of the mat structure; samples were incubated at 55˚C overnight to digest cellular material; RNase was added to the digests and incubated at 37˚C for 1 hour; proteins were precipitated in 7.5M Ammonium Acetate; and nucleic acids were precipitated in isopropanol for 1 hour and then washed in 70% Ethanol and resuspended in 50 µl of TE Buffer.   DNA purity and concentration for each extraction were determined on a Gene_Quantll spectrophotometer (Amersdam Biosciences, Piscataway, NJ). Extractions were stored in TE buffer at -20°C until use. DNA extractions were purified and sequenced by the Research and Testing Laboratories (RTL) (Lubbock, Texas, USA) using massively parallel bacterial tag-encoded FLX amplicon pyrosequences (bTEFAP), as described previously (Dowd et al., 2008a, 2008b) for the V1-V3 region of the 16S rRNA gene. Following sequencing, all failed sequence reads, low quality score sequences (<20), and non-bacterial ribosomal sequences were removed.