Titan one tube rt pcr kit

Total nucleic acids (DNA and RNA) were purified from the filtered samples using a phenol/chloroform/isoamyl alcohol extraction protocol [14 (link)]. The nucleic acids were precipitated with ethanol, and re-suspended in DEPC treated water. Care was taken to ensure nucleic acid pellets from all samples were re-suspended in the same volume and all subsequent steps preserved the initial RNA concentrations. RNA amplifications were performed by first digesting the DNA in the sample with Turbo DNA-free^TM kit (Life technologies; Grand Island, NY) at 37°C for 20 minutes. The sample was then diluted 10⁻⁴ to maintain appropriate template concentrations for the RT-PCR reaction to minimize PCR bias from excessive target molecule concentration [10 (link)]. The diluted rRNA samples were amplified using the Titan One Tube RT-PCR kit (Roche Applied Science, IN) with 16S rRNA universal primers 27 Forward (5' AGA GTT TGA TCC TGG CTC AG 3'; fluorescently labeled with 6-FAM) and 519 Reverse (5' ATT ACC GCG GCT GCT GG 3'). Amplification parameters were 1 cycle at 50°C for reverse transcription at 30 minute, followed by 23 cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 1 minute with a final extension time of 7 minutes. No-RT controls were performed on all samples to assure only RNA was being amplified and profiled. No amplified products were observed in any of the no-RT controls.

The sequencing strategy and primer design to obtain the complete sequence for CbaAr-4005 and 79V-2533 strains were based on a consensus sequence generated from Kern217 (DQ525916.1 and NC_007580.2) and Argentine66 (AY632544.1).
For the 5′UTR amplification and sequencing, a commercial kit (Ambion #AM1700 First Choice RLM Race) was used. For the 3′UTR, the A-Plus Poly (A) Polymerase Tailing Kit (Epicentre Biotechnologies) was used. The manufacturer instructions were applied with the following exceptions: 2.5μl RNAase inhibitor (40U/μl), and 0.5μl A-Plus Poly A (4U/μl) were added and the reaction incubated at 37°C for 10min. The Titan One Tube RT-PCR Kit (Roche) was used for genomic amplification. For sequencing of the amplified fragments the same protocol as that for the 5′UTR was used.
The partial fragments generated for each strain were analyzed and individually selected for assembly to generate a consensus genome sequence using the SeqMan II (v. 5.03) program provided within the LaserGene (DNAStar) package. The complete genome sequences for both strains were submitted to GenBank with the accession numbers FJ753286.2 and FJ753287.2.

Vero cell supernatant was used for RNA viral extraction employing the commercial QIAamp viral RNA MiniSpin Kit (Qiagen). For reverse transcription and PCR, two commercial kits were used, Titan One Tube RT-PCR System (Roche) and Titan One Tube RT-PCR Kit (Roche, Reaction Mix 2), following the manufacturer’s instructions.

All cell lines were treated with 0.2 μM IDR or 0.1 μM Am80 for 4 h. Total RNA was isolated using a high-purity RNA isolation kit (Roche Diagnostics, Mannheim, Germany). Reverse transcription polymerase chain reaction (RT-PCR) was carried out using a Titan One Tube RT-PCR kit (Roche Diagnostics) according to the manufacturer’s instructions. cDNA derived from each cell line was amplified by 28 PCR cycles. The relative signal intensity of bands was determined and standardized using imaging software (Scion, Frederick, MD, USA) as described previously [12 (link), 13 (link)].