Fr10.0103
Fr10.0103 RRL
We followed the genome reduction protocol described in McCormack et al. (22). Briefly, 50-100 ng of DNA was subjected to simultaneous restriction digest (EcoRI and MseI), and sticky-end adaptor ligation. PCR preamplification with EcoRI and MseI primers that targeted the adaptors was performed with Phusion Taq. A second selective amplification was conducted using a fusion reverse primer (consisting of Roche 454 Lib-L primer B and MseI primer) and forward fusion primer made up of Roche 454 Lib-L primer A, sample specific barcode (10 bp) and Eco-RI primer. Following electrophoresis (1.5% agarose gel with 100 bp size ladder), PCR fragments in the range of 400-550 bp were excised and column purified with QIAquick gel extraction kit (Qiagen, Inc., Venlo, Netherlands). The eluted products were cleaned with AMPure XP beads to remove all fragments below the size of 300 bp (Beckman-Coulter). The final products were quantified and assessed for quality before being submitted for 1 x 1/8 PTP sequencing with a Roche 454 GS FLX+ machine at the University of Illinois W.M. Keck Center.
Smithsonian Institution
Fr10.0103