Aux_May_16_euk
NGS sequencing of microorganisms present in a high pH spent nuclear fuel pond
Sequencing of PCR  amplicons of 18S rRNA was conducted with the Illumina MiSeq platform  (Illumina, San Diego, CA, USA) targeting the V9 hyper variable region  (forward primer, 1391F, 5'-GTACACACCGCCCGTC-3'; reverse primer, EukBR,  5'-TGATCCTTCTGCAGGTTCACCTAC-3') for 2250-bp paired-end sequencing. PCR  amplification was performed using Roche FastStart High Fidelity PCR System in  50ul reactions under the following conditions: initial denaturation at 95C  for 2min, followed by 36 cycles of 95C for 30s, 55C for 30s, 72C for 1  min, and a final extension step of 5min at 72C. The PCR products were  purified and normalised to 20ng each using the SequalPrep Normalization Kit .  The PCR amplicons from all samples were pooled in equimolar ratios. The run  was performed using a 4pM sample library spiked with 4pM PhiX to a final  concentration of 12% following the method of Schloss and Kozich.
Aux_May_16_euk
SUB5263014
Aux_May_16_euk