title: 454 of bacteria: pond bacterioplankton and biofilm
design: DNA was extracted using the PowerSoilŽ DNA Isolation Kit (MoBio Laboratories, CA, USA) following the manufacturers instructions using the filter as starting material. DNA concentration of each sample was measured spectrophotometrically (NanoDropŽ ND-1000, Thermo Fisher Scientific Inc., DE, USA) and subsequently standardized over samples. For pyrosequencing, an amplicon library representing all 180 samples was prepared using the eubacterial universal primers E338F and E797R covering the V3 region of the 16S rDNA (Wang and Qian, 2009). We used the composite forward primer 5?-CGTATCGCCTCCCTCGCGCCATCAG MID ACTCCTACGGGAGGCAGCAGT-3? (where the underlined sequence is the 454 Life SciencesŽ primer A-key, MID (multiplex identifier) is a 10bp barcode specific for each sample, and the sequence written in italics is the broad range bacterial primer E338F), and the composite reverse primer 5?-CTATGCGCCTTGCCAGCCCGCTCAG GGGTATCTAATCCTG-?3 (where the underlined sequence is the 454 Life SciencesŽ primer B-key and the sequence written in italics is the broad range bacterial primer E797R). PCR mixture was as follows: 2.5?L 10×PCR buffer (Eurogentec, Seraing, Belgium), 1?L MgCl2 (50Mm; Eurogentec), 2.5?L dNTPs (2mM; Thermo Scientific, MA, USA), 1.0?L of each primer (20pmol/?L each; Eurogentec), 0.2?L Silverstar Taq DNA polymerase (5.0U/ľL; Eurogentec) and 1ľL standardized template DNA (10.74ng/ľL) in a total reaction volume of 25?L. PCR conditions were as follows: 2 min denaturation at 94°C followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C and 1 min elongation at 72 °C, with a final extension at 72°C for 5 min. PCR products were pooled (3xPCR for 1 sample) and separated from contaminants and primer dimers by electrophoresis (1.5-2h at 120V) on a 1.5% agarose gel stained with GelRedTM (Biotium, CA, USA) (10,000×) in 1×TAE buffer. Gel bands were excised and purified using the Qiaquick Gel extraction kit (Qiagen, Venlo, Netherlands). Concentrations of the purified PCR products were determined using Quant-ITTM PicoGreen (Life Technologies, CA, USA) and pooled in equimolar quantities. Pyrosequencing was performed on a Roche GS-FLX with Titanium chemistry according to the manufacturers instructions.