title: DNA 100m
design: 16S gene fragments were amplified by PCR from environmental DNA and cDNA templates using universal primers 926F and 1392R plus adaptors for 454 pyrosequencing. Triplicate PCR reactions (20 µl in triplicates) for each library included: 0.25 Units JumpStart Taq (Sigma), 1x JumpStart PCR buffer, 0.05 µM each primer, 200 µM of each dNTP, and 300 µg/ml BSA.  The PCR reaction was thermocycled: 95°C for 3 min, 25 cycles at 95°C for 30 s, 55°C for 45 s, and 72°C for 90 s followed by a final extension of 10 min at 72°C.  PCR products were separated by electrophoresis on TAE agarose gels and purified using MinElute Gel Extraction Kit (Qiagen).  Final 454 library preparation and sequencing was conducted at the Joint Genome Institute (JGI) in Walnut Creek, CA.