title: Bacterial microbiome (PGM) of domestics dog: faecal sample: Sample DOG24
design: "Total genomic DNA (gDNA) was isolated from approximately 250 mg of frozen (-20oC) faecal samples using the the PowerSoil Kit (Mo-Bio) utilising a FastPrep-24 Instrument and setting 6 for 40 s 24 (MP Biochemicals). Extracted DNA was eluted into 100 ul of ddH20 water and stored at -20oC. Microbiome analysis was performed using PCR primers amplifying 16S rRNA gene V4 variable region; 16s-515F (GTG CCA GCM GCC GCG GTA A) and 16s-806R (GGA CTA CVS GGG TAT CTA AT) (Caporaso et al. 2011). A single-step PCR with the HotStarTaq Plus Master Mix Kit (Qiagen, USA) included 94oC for 3 minutes, followed by 28 cycles of 94oC for 30 seconds, 53oC for 40 seconds and 72oC for 1 minute and a final step at 72oC for 5 minutes. Amplicon diversity sequencing services (bTEFAP) on an Ion Torrent PGM (Life Sciences) was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA)."