title: Archaea 16S tag-sequencing Laptev Sea 2007 station VB3807 6m
design: "DNA was extracted from fractionated microbial biomass (small fraction 0.2 - 3 micrometers).  Library was produced by amplifying archaeal V3-V5 region of the 16S rRNA gene.  Libraries was generated by first amplifying V2-V5 region with the A109F (5'-ACKGCTCAGTAACACGT-3') and A915R (5'-GTGCTCCCCCGCCAATTCCT-3') primers.  Reactions were performed in a volume of 25 microleters containing: 1X HF buffer, 0.2 mM dNTP mix, 0.2 micrometers of each primer and 0.5 units of Phusion Polymerase.  PCR reactions were performed in triplicates containing different template concentrations.  Thermocycler was set as follows: initial denaturation at 98 degrees Celcius for 30 seconds followed by 35 cycles of denaturation at 98 degrees Celcius for 10s, annealing at 50 degrees Celcius for 30 seconds and 72 degrees Celcius for 30 seconds and a final elongation at 72 degrees Celcius for 5 minutes.  A final amplification of 10 cycles was performed on the first PCR product with the primers A340f (5'-CCCTAHGGGGYGCASCA-3') containing the barcodes and the 454 adapter A and the A915r (5'-GWGCYCCCCCGYCAATTC-3') containing the 454 adapter B.  PCR conditions were identical as described previously, except for the annealing temperature which was set at 52 degrees Celcius.  Final PCR products were purified with a PCR purification kit (Feldan).  Libraries were pooled by series of maximum 12 for 454 sequencing."