DNeasy UltraClean Microbial Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

DNA Extractions and Sequencing
Isolates were grown to late stationary phase in 5 ml of media. Cultures were centrifuged for 10 min at 4000 rpm to generate a cell pellet. The cell pellet for each isolate was re-suspended in 500 μl dHO and DNA was extracted using the UltraClean Microbial DNA Isolation Kit from MoBIO Laboratories, Inc. (Carlsbad, CA, USA). Full-length 16S rRNA amplicons were generated with bacterial primers 8F/1492R () with the following PCR protocol; 94°C for 2 min, (94°C for 30 s, 59°C for 1 min, 68°C for 2 min) × 30 cycles, 68°C for 5 min, then cloned with the TOPO TA Cloning Kit for Sequencing (Life Technologies Inc., Grand Island, NY, USA). Ten clones per isolate were picked and sent for Sanger sequencing at GeneWiz Inc., (South Plainfield, NJ, USA) to confirm culture purity. Full length 16S rRNA DNAs are deposited under NCBI accession numbers KU707917–KU707919. Genomic DNA was sent to the United States Department of Energy’s Joint Genome Institute for full genome sequencing. Isolate genomes and raw sequencing data are available from the GOLD database via accession numbers: NP_8HT: Gp0114906, NP_1H: Gp0115197, sp. NP_8HO: Gp0114905 and under NCBI project ID: 303658. For total microbial community analysis at both depths (4 and 68 mbsf), 10 g of sediment from each depth was processed with the PowerMax Soil DNA Isolation Kit from MoBio Laboratories, Inc. (Carlsbad, CA, USA). Total bacterial community was amplified from 2 ng template DNA using 16S rRNA primers 27F/338R via PCR protocol: 95°C for 2 min, (95°C for 30 s, 60°C for 1 min, 72°C for 2 min) × 35 cycles, 72°C for 5 min. An extraction blank was processed with the samples. All PCR products, including the one from the extraction blank, were sent for Illumina library prep and sequencing at Next Generation Sequencing Services (Shallowater, TX, USA). This involved a nested PCR approach, resulting in a minimal number of cycles occurring after initial PCR amplification, which may slightly reduce the diversity of the environmental samples. Amplicon sequencing data is available under NCBI accession numbers SRS1277883–SRS1277884.

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Manufacturer protocol

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