Phusion high fidelity pcr kit

Genomic DNA was extracted from blood or muscle of 18 different Iberian lynxes using standard phenol-chloroform methods [35 ] and then the three amplification strategies were tested on the same extract for each individual. We used the Universal Tailed Amplicon Sequencing design by Roche consisting of a two-round PCR approach in which the first round amplifies the target locus using primers with a universal 5’ extension and the second adds 454 sequencing adapters and an individual tag to the amplicons generated in the first PCR. Amplicons were tagged so that sequences could be sorted by sample and strategy (i.e. the four amplicons per individual generated using pooled-PCRs strategy were given the same tag). Artefact formation during PCR was minimized by using the Phusion^® High-Fidelity PCR Kit by Roche, which reduces nucleotide misincorporation [36 (link)], and by implementing long extension times and no final extension step, which should prevent chimera formation. PCRs were run in a final volume of 10 μl following manufacturer indications. Cycling conditions were the same for all PCRs: an initial denaturation at 98° for 30 sec and 25 cycles of 98°C for 10 sec, 57°C for 30 sec and 72°C for 2 min. PCR products were pooled and sequenced on a 454 GS Junior system.

Genomic DNA was extracted from blood or tissue using standard phenol-chloroform methods [81 ]. To produce sequencing libraries, we used the Universal Tailed Amplicon Sequencing design by Roche consisting of a two-round PCR approach in which the first round amplifies the target loci using primers with a universal 5′ extension and the second adds 454 sequencing adapters and a sequence tag to the amplicons generated in the first PCR to unambiguously identify each sample. Artefact formation during PCR was minimized by using the Phusion High-Fidelity PCR Kit by Roche, which reduces nucleotide misincorporation [82 (link)], and by implementing long extension times and no final extension step, which prevents chimera formation. PCRs were run in a final volume of 10 μl following manufacturer indications. Cycling conditions were the same for all PCRs: an initial denaturation at 98° for 30 s and 25 cycles of 98 °C for 10 s, 57 °C for 30 s and 72 °C for 2 min. PCR products were equimolarly pooled and sequenced on a 454 GS Junior system.