title: 16S amplification of mus musculus: gut metagenome: Sample Day8.Vancomycin.Cohort3.Feces.Sample21
design: The DNA extracted from fecal samples was then subjected to rtPCR of 16S RNA gene sequences. We used the broad range bacterial 16S primers 517F (5’- GCCAGCAGCCGCGCTAA -3’) and 798R (5’- AGGGTATCTAATCCT -3’) at 0.2 mM concentrations with the DyNAmo SYBR green qPCR kit (Finnzymes). Sample amplification was compared to standard curves to quantify 16S copy number. Cycling conditions were as follows: 95?C for 15 minutes, then 40 cycles of 94?C for 15 seconds, 52?C for 30 seconds, and 72?C for 30 seconds. The program finished with 95?C for 15 minutes, 60?C for 1 minute, and 95?C for 15 minutes.Replicate PCRs were pooled, and amplicons were purified using the Qiaquick PCR Purification Kit (Qiagen). PCR products were quantified and pooled at equimolar amounts before Illumina barcodes and adaptors were ligated on using the Illumina TruSeq Sample Preparation protocol. The completed library was sequenced on an Ilumina Miseq platform following the Illumina recommended procedures. 