SSU rRNA Gene Eukarya V9 amplification of sample: DSE_0004_2008_06_20
Genomic Eukarya DNA of the SSU rRNA gene V9 hypervariable region was amplified using forward primer 1380F (CCCTGCCHTTTGTACACAC) and primer 1389F (TTGTACACACCGCCC) and reverse primer 1510R (CCTTCNGCAGGTTCACCTAC). We generated PCR amplicons in triplicate 30 uL reaction volumes with an amplification cocktail containing 1.0 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase Mix (Pierce Nucleic Acid Technologies, Milwaukee, WI), 1.5 mM MgSO4 and 0.2 uM 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 94C, 3 minute denaturation step was followed by 30 cycles of 94C for 30s, 57C for 60s, and 72C for 90s before a final 10 minute extension at 72C.  The triplicate PCR products were pooled after amplification, purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in 12 uL 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.