title: SSU rRNA Gene Eukaryal V9 Amplification with 1380 and 1389 forward primers
design: Genomic DNA was amplified in 2 separate PCR experiments; one employing the primer set 1380F/1510R and the other using the primer set 1389F/1510R. We generated PCR amplicons in triplicate 30 µL reaction volumes for each experiment with an amplification cocktail containing 1.0 U Platinum Taq Hi-Fidelity Polymerase (Strategene, La Jolla, CA), 1 X Hi-Fidelity buffer, 200 µM dNTP PurePeak DNA polymerase Mix (Pierce Nucleic Acid Technologies, Milwaukee, WI), 1.5 mM MgSO4 and 0.2 µM of each primer. We added a total of 10 ng template DNA to each PCR reaction and ran a negative, no-template control for each primer pair.  Each replicate was assigned a unique forward primer incorporating the 1380 or 1389 primer sequence, the 454 Life Science Adaptor A, and the distinct 5-base key to allow us to bioinformatically separate information from each sample after sequencing as described in Huber et al., 2007.  The two forward primers were not multiplexed in a single reaction, but amplified separately for each of the 16 samples, necessitating 32 total reaction cocktails, each with a unique barcoded forward primer.  We ran a separate no-template negative control for each unique barcoded primer.  Amplification conditions described in Sogin et al., 2006 were modified as follows: the initial 94 degreeC, 3 minute denaturation step was followed by 30 cycles of94 degreeC for 30 s, 57°C for 60 s, and 72° for 90 s before a final 10 minute extension at 72° C.  The triplicate PCR products were pooled after amplification, purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in 12 µL of Qiagen buffer EB following the manufacture's protocol.  We assessed the quality, size and concentration of PCR products on a Bioanalyzer 2100 (Agilent, Palo Alto, CA) using a DNA 1000 Lab Chip.