title: Biopurification system IncP-1 trfA 3.3- d
design: "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland).    Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139.  Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene"