16S sequencing of distal duodenum
DNA was isolated from intestinal fecal content regions using the QIAamp DNA Stool Isolation kit and Qiacube from Qiagen per manufacturers recommended protocol.  A 16S rRNA library was prepared as follows.  The PCR reaction contained microbial genomic DNA (5ng/ul in 10mM Tris, pH 8.5), 1 M 16S Amplicon Forward primer (5- TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3), 1 M 16S Amplicon Reverse primer (5- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3), 2x KAPA HiFi HotStart ReadyMix (Roche Kapa Biosystems, Wilmington, MA).  PCR program parameters was initial denaturation at 95C for 3 min, 25 cycles of 95C for 30 sec, 55C for 30 sec, and 72C for 30 sec and then a final extension of 72C for 5 min.  Next, the Index PCR was prepared using the Nextera XT Index Kit, PCR Clean-up, and Illumina MiSeq sequencing was performed following manufacturers protocol (Illumina, San Diego, CA).
Anastomosis.doudenum.rep8