Superscript 3 one step rt pcr system with platinum taq high fidelity kit

Both complete and partial coding nucleotide sequences were determined using NGS methods: overlapping amplicons spanning either the complete genome sequence or the first part of the genome were produced from the extracted RNA (see corresponding section) using the SuperScript® III One-Step RT-PCR System with Platinum®Taq High Fidelity kit (Invitrogen) and specific primers (detailed in the supplementary Table S3). Sequencing was performed using the PGM Ion torrent technology (Thermo Fisher Scientific) following the manufacturer’s instructions. Consensus sequence determination was done using CLC genomics workbench software (CLC bio-Qiagen). Substitutions with a frequency higher than 5% were considered for the analysis of intra-population genetic diversity and major/minor variants identification (minor variants: variant frequency >5% and ≤75%; major variants: variant frequency >75%).

In 1987, PBMC from animal Mf186-76 were co-culture with H9 cells. A vial containing cell supernatant from this co-culture was obtained (a gift from R.C. Desrosiers). Viral RNA was isolated using the High Pure Viral RNA Kit (Roche USA, Indianapolis, IN). Amplicon-specific cDNAs were produced using the SuperScript^™ III One-Step RT-PCR System with Platinum^® Taq High Fidelity kit (Invitrogen, Carlsbad CA) using primers listed in S1 Table; amplicons were either sequenced directly, or TA-cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and then sequenced. The sequence of the full-length SIVmfa genome was deposited in GenBank under the accession number KU500642. The SIVmfa vif sequence was previously reported under KF030930 [22 (link)].