Dataset: coral-microbe T-RF - spawning
Deployment: Rappe_2006

T-RFLP analysis of bacterial SSU rRNA genes. Approximately 1 l of seawater was filtered through a GF/A glass microfiber membrane pre-filter (1.6 µm nominal pore size, Whatman International) followed by a 13 mm diameter, 0.2 µm pore-sized polyethersulfone membrane (Supor 200, Pall Gelman). Filters were stored at -80°C in DNA lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 1.2% v/v Triton X100) (Suzuki et al. 2001). Genomic DNA was extracted from the 0.2 µm pore-sized polyethersulfone membranes using a modified version of the DNeasy Tissue kit (Qiagen) (Becker et al. 2007), and quantified using a PicoGreen fluorescent assay (Invitrogen) on a SpectraMax M2 plate reader (Molecular Devices). For terminal restriction fragment length polymorphism (T-RFLP) analysis (Liu et al. 1997), bacterioplankton small-subunit (SSU) ribosomal RNA (rRNA) genes (including those of heterotrophic bacteria, cyanobacteria, and eukaryotic plastids) were first amplified via the polymerase chain reaction (PCR) using the bacterial primers 27F-B-FAM (5’-AGRGTTYGATYM TGGCTCAG-3’) and 519R (5’-GWATTACCGCGGCKGCTG- 3’), with ‘FAM’ indicating 5’ end-labeling of the 27F-B primer with the 6-carboxyfluorescein (FAM) fluorochrome. Each 50 µl PCR reaction contained 0.625 U of PicoMaxx high-fidelity DNA polymerase (Stratagene), 1× PicoMaxx reaction buffer, 200 µM of each deoxynucleoside triphosphate (dNTP), 200 nM of each primer and 10 ng of environmental genomic DNA template. After an initial denaturation step at 95°C for 5 min, the reaction conditions were: 24 cycles of 95°C denaturation for 30 s, 55°C annealing for 1 min, and 72°C extension for 2 min, concluding with an extension at 72°C for 20 min. The reactions were performed in a MyCycler Personal Thermal Cycler (Bio-Rad Laboratories). Amplification products were purified using the QIAquick PCR Purification Kit (Qiagen), and subsequently restricted in a 10 µl reaction containing 100 ng of purified amplification product, 2 µg of bovine serum albumin (BSA), 1× enzymatic reaction buffer, and 5 units of HaeIII restriction endonuclease (Promega) for 7 h at 37°C. Restriction digests were purified using the QIAquick Nucleotide Removal Kit (Qiagen), and 30 ng µl-1 of each product was subsequently electrophoresed on an ABI 3100 Genetic Analyzer (Applied Biosystems). GeneMapper software (Applied Biosystems) was used to estimate the length (in base pairs) and relative abundance of the resulting fragments. Operational taxonomic units (OTUs) were identified as terminal restriction fragments (T-RFs) detected between 33 and 550 bp in length. To account for small differences in the amount of DNA loaded on the ABI 3100, data were normalized by excluding peaks that contributed to <0.05% of the total peak area for each sample (Sait et al. 2003). An average of 2 peaks were removed from the samples, with a minimum of no peaks and a maximum of 19 peaks removed. An in-house dataset linking cloned bacterial and plastid SSU rRNA gene sequences to T-RFLP profiles from Kaneohe Bay seawater was analyzed in order to putatively identify T-RFs of interest (Yeo et al. unpubl.).