title: SSU rRNA Gene Eukaryal V9 Amplification with 1389 forward primer
design: Genomic DNA from MHB was amplified in employing the primer sets 1389F/1510R. We generated PCR amplicons in triplicate 30 µL reaction volumes 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.  Amplification conditions described in Sogin et al., 2006 were modified as follows: the initial 94° C, 3 minute denaturation step was followed by 30 cycles of 94°C 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 manufacturer’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.