16s rRNA sequencing of Juturnia kosteri collected at bitter lake site U7: replicate 1
Library construction used the ~250 bp V4 fragment of the 16S rRNA gene as amplified with 515f/806r single sided GOLAY barcoded primers. PCR was performed in triplicate using 10 L 2.5x 5 PRIME HotMasterMix (Quantabio, Beverly, MA), 0.5 L 10 M forward primer, 0.5 L 10 M reverse primer (with unique GOLAY barcode), 1 L of gDNA (~20 ng) template in  a total reaction volume of 25 L. Thermal cycling conditions were as follows: 94C for 3 min, (94C for 25s, 50C for 60s, 72C for 90s) for 35 cycles, 72C for 10 min polish performed on a PTC-200 MJ Research thermocycler.  The target amplicon size (~350 bp) was confirmed by gel electrophoresis on a 1% agarose gel. Libraries were quantified using a SYBER Green I Assay and 240 ng of each library was pooled and purified using a MoBio UltraClean PCR Clean-Up Kit. Finally, the library pool was quantified by qPCR using the KAPA Universal Illumina Quantitation Kit (Roche KK4824).  Libraries were prepared for loading following Illuminas MiSeq System Denature and Dilute Libraries Guide (Illumina 15039740 v01). Sequencing was performed on an Illumina MiSeq Benchtop Sequencer 300-cycle v2 kit 2x150PE with a 10% spike-in of PhiX v3 control at Miami Universitys Center for Bioinformatics and Functional Genomics.
SUB10022451
JK-BLU7-1