Orbicella faveolata sample collected in Mar-11 Mar.Mf378.B: Sample Mar.Mf378.B
"Samples of G. ventalina and O. faveolata were collected in Puerto Rico. Each sample was placed in a sterilized bag, stored on ice for transport to the lab, and drained of excess seawater before flash-freezing in liquid nitrogen and shipping on dry ice. We extracted microbial DNA from whole organism samples of both coral species. 1x2 cm of frozen tissue from the whole organism was ground to a powder in liquid nitrogen with a sterilized mortar and pestle. The optimal starting quantities were 100 mg of G. ventalina and 200 mg of O. faveolata. We added the samples to PowerSoil DNA Isolation Kit (MoBio Laboratories) bead tubes, then stored them at -80oC. We modified the kit protocol to improve quality and yield as per Sunagawa et al., 2010: 1) incubation with 10 U uL-1 lysozyme (0.19uL; Epicentre) for 10 min at room temperature; 2) incubation with ~0.8 mg mL-1 proteinase K (25uL; Invitrogen) for 60 min at 65oC; 3) addition of 400 mg each of 0.1 and 0.5 mm zirconia/silica beads; and 4) homogenization for 30 s at 30 beats/s (Retsch MM300 Bead Beater). Total genomic DNA concentrations were determined using the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen). Approximately 14 ng (~1-5 ul) of template DNA was used in each of three replicate, 30 ul PCR reactions with Taq DNA Polymerase (Invitrogen). The reactions were pooled and run on a 2% Agarose-TAE gel to confirm products. The pooled products were then purified with Ampure magnetic purification beads (Agencourt) and quantified using the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen). Pooled samples were sent to Engencore (University of South Carolina) for pyrosequencing using Roche 454 with Titanium and FLX platforms."