alias: H7LY04103_Flx1304.Snottite16_D1A_E1_TCACGTACTA.sff.Snottite16_D1A_E1_TCACGTACTA
title: Snottite
design: For DNA extraction, ~300 µl biofilm slurry was suspended in 650 µL PTN buffer (120 mM Na2HPO4, 125 mM Tris, 0.25 mM NaCl [pH 8]) and incubated at 37°C for 15 min with 40 µl lysozyme (50 mg ml-1 in TE) and 10 µl proteinase K (10 mg ml-1 in TE). After the addition of 150 µl 20% (wt/v) sodium dodecyl sulfate, incubation was continued for 15 min at 65°C with shaking at 500 rpm. The sediments were bead beaten (45 s at 6.5 ms-1 in a FastPrep-24 (MP Biomedicals, Solon, OH) with ,0.2 ml of zirconia-silica beads (1:1 mix of 0.1- and 0.7-mm diameter; Roth, Karlsruhe, Germany) and 100 µl of phenol- chloroform-isoamyl alcohol (25:24:1) in 2-ml screw-cap vials. Afterwards nucleic acids were sequentially purified by extraction with 1 volume of phenol-chloroform-isoamyl alcohol (25:24:1) and 1 volume of chloroform-isoamyl alcohol (24:1). Purified nucleic acid was then precipitated with 2 volumes of 30% polyethylene glycol by incubation at 4°C for 12 h and subsequently centrifuged at 20,000 g and 20°C for 30 min. All used chemicals were from Sigma-Aldrich, St. Louis, MO, if not otherwise stated. For each biological sample, two parallel extractions were pooled and stored at -20°C until further analysis.   Amplicon pyrosequencing was performed on a 454 GS FLX Titanium system (Roche, Penzberg, Germany). Barcoded amplicons for multiplexing were prepared using the primers Ba27f and Ba519r extended with the respective A or B adapters, key sequence and multiplex identifiers (MID) as recommended by Roche (USA). Pyrotag PCR was performed in a Mastercycler ep gradient (Eppendorf, Hamburg, Germany) with the following cycling conditions: initial denaturation (94°C, 5 min), followed by 28 cycles of denaturation (94°C, 30 s), annealing (52°C, 30 s) and elongation (70°C, 60 s). Each 50 ml PCR reaction contained 16 PCR buffer, 1.5 mM MgCl2, 0.1 mM dNTPs, 1.25 U recombinant Taq polymerase (Fermentas, St. Leon-Rot, Germany), 0.2 mg ml-1 bovine serum albumin (Roche), 0.3 mM of each MID-primer (Biomers, Ulm, Germany) and 1 µl of template DNA. Amplicons were purified using PCRExcract mini kit columns (5prime, Germany) and pooled in an equimolar ratio of 10^9 molecules ml-1 as quantified by the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen, Paisley, UK). Emulsion PCR, emulsion breaking and sequencing were performed applying the GS FLX Titanium chemistry following supplier protocols.
identifiers.namespace: SUB1646831
identifiers.id: H7LY04103_Flx1304.Snottite16_D1A_E1_TCACGTACTA.sff.Snottite16_D1A_E1_TCACGTACTA