title: Dietary DNA metabarcoding of small-herbivore fecal samples
design: We amplified the P6 loop of the chloroplast trnL (UAA) intron. We performed PCR in 25 L reactions using 1.5 mM MgCl2, 200 M each dNTP, 0.2 M each primer [trnL(UAA)c/trnL(UAA)h], Platinum Taq DNA polymerase, and 2 L of DNA extract. The g and h primers were modified to contain an adapter overhang. Thermocycling followed a program of initial denaturation at 95C for 5 min, followed by 35 cycles of 95C for 30 s, 55C for 30 s, and 72C for 1 min, with a 10 min final extension at 72C. We verified PCR success by running 3 l of PCR product on a 1.5% agarose gel. Initial amplicons were then sent to the University of Rhode Island sequencing core where multiplex identification (MID) tags and P5/P7 adapters were annealed to the overhang adapter in a second-round PCR to enable Nextera Illumina sequencing. Libraries were pooled at equimolar concentrations and sequenced on the Illumina MiSeq platform with 250 x 250 bp paired-end chemistry.
identifiers.namespace: SUB15713812
identifiers.id: FJ20220489