Soil microbiome from non-contaminated control near the 1975 Oil Spill Site Rep 1
DNA was extracted from 0.5 g of soil, using an Exgene soil DNA minikit from GeneAll (Seoul, Korea), using 550 l buffer SL (extraction buffer), 50 l buffer RH, 300 l buffer PD, 900 l buffer TB (tissue binding), 500 l buffer NW (wash buffer N), and 50 l elution buffer. Centrifugation was carried out between each extraction step on a 5810R Eppendorf centrifuge.  Polymerase chain reaction (PCR): Partial sequences of the bacterial 16s ribosomal RNA (rRNA) gene region from the extracted DNA were amplified by PCR. The primers used for the 16s rRNA gene were 27F [5'-AGAGTTTGATCCTGGCTCAG-3'] and 1100R [5'-GGGTTGCGCTCGTTG-3'] (Lane 1991). PCR was carried out on an Applied BiosystemsVeriti 96-Well Thermal Cycler. The 25 l reaction mix used for the PCR runs consisted of 12.5 L HS Taq Mix Red (PCR Biosystems, London, UK), 9.5 L ultrapure water, 1.0 L extracted DNA, 1 L forward and reverse primers.The PCR conditions used were 3 m at 98C, 20 cycles of 30 s at 98C, 30 s at 55C, and 60 s at 72C, followed by 5 m at 72C. The final PCR products were detection was conducted by Hylabs Inc. by using the Fluidigm Access Array primers for Illumina to generate libraries compatible for sequencing on the Miseq. Samples were measured for concentration by Qubit and size by Tape Station and then sequenced on the illumine Miseq using a Miseq V2 sequencing kit (500 cycles) to generate 2x250 paired end reads.