Exonuclease 1 shrimp alkaline phosphatase exosap it enzyme

Viral RNA was extracted from culture supernatants using the QIAamp Viral Mini Kit (Qiagen CA, USA) according to the manufacturer's instructions. The complete HN gene was amplified by RT-PCR using three sets of primers previously described [1 (link)].
The PCR amplicons were electrophoresed on a 2% agarose gel (Sigma-Aldrich Co., USA) stained in 2 μg/ml ethidium bromide (Sigma-Aldrich Co., USA) solution and visualized using the E-box gel documentation system (Vilber Lourmat, France). The amplicons were subsequently purified using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, USA) and sequenced on both strands on an automated 3500xL Genetic Analyzer (Applied Biosystems, USA) using the same primers. Cycle sequencing was performed using the Big Dye Terminator Cycle sequencing kit v3.1 (Applied Biosystems, USA).

A 440 bp portion of the intergenic region of ORF1a and ORF1b of the RNA-dependent RNA polymerase (pol) gene of HCoVs was amplified by RT-PCR following the method described by Woo et al. [17 (link)] . RT was performed using the SuperScript III kit (Invitrogen, San Diego, USA) following manufacturer’s instructions. The forward primer was 5′-GGTTGGGACTATCCTAAGTGTGA-3′ whereas the reverse primer was 5′ -CCATCATCAGATAGAATCATCATA-3′. The PCR mixture (50 μl) contained RNA (300 ng), PCR buffer containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 3 mM MgCl₂, 0.01 % gelatin, 200 μM each of deoxynucleoside triphosphates, and 1.0 U of Taq polymerase (Boehringer, Mannheim, Germany). The amplification was carried out as described by Woo et al. [17 (link)]. using the ABI 9700 automated thermal cycler (Applied Biosystems, Foster city, USA). The PCR amplicons were purified using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, California, USA) and sequenced directly on both strands with the same primers used in the PCR on an automated ABI 3500XL Genetic Analyzer (Applied Biosystems, Foster city, USA). Cycle sequencing was performed using the Big Dye Terminator v3.1 sequencing kit (Applied Biosystems, Foster city, USA) according to manufacturer’s instructions.

Viral RNA was extracted from infected culture supernatants with a QIAamp Viral Mini Kit (Qiagen, Inc., USA), according to the manufacturer’s instructions. Partial VP1 gene (3′-end of VP1 gene) was amplified by RT-PCR using primers 292 (5′-MIGCIGYIGARACNGG-3′, position: 2612–2627) and 222 (5′-CICCIGGIGGIAYRWACAT-3′, position: 2969–2951) as previously described (Oberste et al. 2006 (link); Vignuzzi et al. 2005 (link)). PCR amplicons (~350 bp) were analyzed by electrophoresis using 1.0 % Agarose gels (Sigma-Aldrich Co., USA), stained in ethidium bromide (0.5 μg/ml) and visualized using the Alpha Imager (Alpha Innotech, USA). Amplicons were purified using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, USA) and sequenced directly in both directions using the RT-PCR primers. Sequencing was performed using Big Dye Terminator Cycle sequencing kit v3.1 (Applied Biosystems, USA) and analyzed using the automated 3500xL Genetic Analyzer (Applied Biosystems, USA) according to the manufacturer’s instructions.