Collstrup Cu polluted soil (C) U-linker amplified 16S rRNA gene
"Library construction was done in two steps: one PCR to amplify the gene region of interest, and a second PCR to add 454 FLX Titanium adapters and MID sequences. Primers in the first PCR had U-linkers (U1: CAGATACACCGTGCCATACC, U2: CTGGAGCCTAATTCATCACG) fused to the 5'-end while primers in the second PCR had U-linkers fused the 3'-end. The 16S primers were (F: U1-CCTAYGGGRBGCASCAG, R: U2-GGACTACNNGGGTATCTAAT)( Hansen et al. 2012. Diabetologia. 55, 2285-2294). PCR amplifications were 25 µl reaction mixtures consisting 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) and 1 µl template DNA diluted to 5-10 ng/µl. First round PCR conditions were 98 deg C for 30 s, 35 cycles of 98 deg C for 10 s, 56 deg C 30 s, 72 deg C for 30 s, with a final 5 min extension at 72  deg C. After PCR, samples were held at 70 deg C for 3 min and then kept on ice until visualized by gel electrophoresis on a 1% (w/v) agarose gel with ethedium bromide. Product specific bands were cut out and DNA purified with the Montage Gel Extraction Kit (Millipore, Billerica, MA, USA). 1 µl purified DNA was used in the second round PCR as described above but with 15 cycles and the U-linker Ta of 58 deg C. Amplicons from the same sample were given the same MID. I.e., for each sample there were 5 amplicons with different primer sequences but identical MID. The amplicons with adapters and MIDs were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). Approximately equal concentrations of tagged PCR products from each sample were used to ensure near-equal sample representation. Sequencing was done at The Danish National High-Throughput DNA-Sequencing Centre on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland)."