Qiaquick 96 well pcr purification kit

PCR was performed using universal primers flanking the variable 4 (V4) region of the bacterial 16S rRNA gene [76 (link)]. Genomic DNA samples were amplified in duplicate. Each reaction contained 10–30 ng genomic DNA, 10 μM each primer, 12.5 μl 2x HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to a final reaction volume of 25 μl. PCR was carried out under the following conditions: initial denaturation for 3 min at 95°C, followed by 25 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 55°C and elongation for 30 s at 72°C, and a final elongation step for 5 min at 72°C. PCR products were purified with the QIAquick 96-well PCR Purification Kit (Qiagen, Germantown, MD, USA) and quantified using Qubit dsDNA HS Assay kit (Invitrogen, Oregon, USA). Samples were equimolar pooled and sequenced by the University of Wisconsin–Madison Biotechnology Center with the MiSeq 2x250 v2 kit (Illumina, San Diego, CA, USA) using custom sequencing primers.

Genomic DNA was extracted from undiluted fecal aliquots using a bead-beating protocol [16 (link)]. Briefly, feces was re-suspended in a solution containing 500 μL of extraction buffer (200 mM Tris (pH 8.0), 200 mM NaCL, 20 mM EDTA), 210 μL of 20% SDS, 500 μL phenol:chloroform:isoamyl alcohol (pH 7.9, 25:24:1, Invitrogen, Carlsbad, CA), and 500 μL of 0.1 mm diameter zirconia/silica beads. Samples were mechanically disrupted using a bead beater (BioSpec Products, Barlesville, OK; maximum setting for 3 min at room temperature), followed by centrifugation, recovery of the aqueous phase, and precipitation with sodium acetate and isopropanol. QIAquick 96-well PCR Purification Kit (Qiagen, Germantown, MD, USA) was used to remove contaminants. Isolated DNA was eluted in Tris-EDTA buffer and stored at −80 °C until further use.

Total genomic DNA for use in 16S rDNA sequencing, DNA microarray profiling, MLST, SCCmec typing and dru typing was extracted using the StaphyType kit (Alere Technologies GmbH, Jena, Germany) according to the manufacturer’s instructions. Apart from PCR for DNA microarray profiling, all PCRs were performed using GoTaq DNA polymerase (Promega, WI, USA). PCR products were purified using the GenElute PCR clean-up kit (Sigma, Wicklow, Republic of Ireland) or, for MLST, the QIAquick 96 well PCR purification kit (Qiagen, Crawley, UK). All DNA sequencing reactions were carried out commercially by Source BioScience LifeSciences (Waterford, Republic of Ireland).