title: 16S Illumina seqs of Scaptotrigona sp.:whole abdomens
design: Prepared amplicon libraries using the 799F (CMGGATTAGATACCCKGG)-1115R (AGGGTTGCGCTCGTTG)16S rRNA gene primers. Briefly, we first included the partial Illumina sequencing adapter and a unique combination of 8-mer barcodes for each sample with primers that include the genomic primer sequence. We performed 25 cycles of PCR in 25 L reaction with a 52 C annealing temperature using Phusion MasterMix (Thermo Scientific, Waltham, MA). To clean these reactions, we used Exonuclease and Shrimp Alkaline Phosphatase to remove excess primers and DNTPs, respectively. We then performed a second PCR with 1 L of cleaned PCR product as template and primers that complete the Illumina sequencing construct: PCR2F (CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCC TGC) and PCR2R (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG). To normalize the amount of DNA in each library, we used SequalPrep normalization plates (Invitrogen, Waltham, MA), following the manufacturers protocol. We used 5 L of each library to create a library pool, which we then cleaned with Ampure XP beads (Beckman Coulter, Brea CA) to remove primer-dimer and excess master mix components.