Kingfisher flex dna extraction robot

The liquid effluent (15 mL) from each reactor was collected weekly and stored at − 20 °C prior to DNA extraction. To obtain DNA for 16S amplicon sequencing, 1 mL of the liquid effluent was centrifuged and the pellet was used for DNA extraction. The template DNA was extracted using the PowerMag Soil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) on a KingFisher Flex DNA extraction robot (Thermo Fisher Scientific, Waltham, MA, USA). The DNA extraction was performed following the manufacturer protocol except that samples were subjected to bead beating at maximum intensity on a FastPrep-96 Homogenizer (MP Biomedicals LLC., Santa Ana, CA, USA) for 4 pulses of 30 s each and a 5-min interval between pulses. DNA template concentration was determined using a Qubit Fluorometer (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA), and these reactor digest DNA extracts were stored at − 20 °C until further use.

Fecal samples were thawed on ice and ~ 200 mg from each sample was added to sterile water at a ratio of 1:3. Homogenization involved bead beating using a MagNaLyser (Roche, Basel, Switzerland) twice at 6500 rpm for 20 s with 1 minute cooling at 4°C between runs as described previously [30 (link)]. DNA was extracted using the Mag Mini LGC kit (LGC Genomics, Hoddesdon, UK) according to the manufacturer's recommendations using a KingFisher Flex DNA extraction robot (Thermo Fisher Scientific, Waltham, MA, USA). Adequate DNA quality and quantity in samples were ensured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). DNA samples were stored at -20°C until processing.

Right after dissection, fecal samples were collected from the colon and placed in 400 μL S.T.A.R buffer (Roche, USA) with acid-washed glass lysing beads (approximately 0.2g <106 μm, 0.2g of 425–600 μm and 2–4 beads of 2mm Sigma-Aldrich) and stored at -80°C for further processing. All samples were homogenized twice on FastPrep 96 (1,800 rpm, 40 sec, 5 min cooling step in-between, MP BioMedicals). Processed samples were then centrifuged (13,000 rpm, 10 min) and 50 μL supernatant was transferred to 96-well plates for protease treatment and DNA extraction using Mag Mini LGC kit (LGC Genomics, UK) on KingFisher Flex DNA extraction robot (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.