High fidelity one step rt pcr kit

The rescued rSVA-eGFP was serially passaged in BSR cells fifteen times. Eight BSR cell monolayers were inoculated with the P1, P3, P5, P7, P9, P11, P13 and P15 rSVA-eGFPs, respectively, and then incubated at 37 °C for 24 h. Fluorescence intensities corresponding to their progenies (P2, P4, P6, P8, P10, P12, P14 and P16) were observed under the fluorescence microscope. The culture supernatants at P6, P8, P10, P12, P14 and P16 were harvested to extract viral RNAs for RT-PCR analysis using the F1/R1 primer pair, as described in Subheading 2.4.1. In order to analyze the foreign sequence in the rSVA-eGFP genome, another primer pair (F2/R2, Table 1 and Figure 1A) was designed for amplifying a 1004-bp fragment by RT-PCR using the High Fidelity One Step RT-PCR Kit (Takara, Dalian, China). The RT-PCR reaction underwent 45 °C for 10 min, 94 °C for 2 min and then 30 cycles at 98 °C (10 s), 55 °C (15 s) and 68 °C (10 s). All F1/R1- and F2/R2-amplified products were detected by agarose gel electrophoresis, and the latter was subjected to Sanger sequencing.

The culture supernatant from the BHK-21cells of rSVA-p16 at passage 3, 6, 10, and 15 (P3, 6, 10, and 15) was harvested for extracting viral RNA by a Viral RNA/DNA Extraction Kit (Takara, Dalian, China). The viral RNAs of P3, P6, P10, and P15 virus stocks were extracted and reverse transcribed into cDNA. The extracted RNA was used as a template for RT-PCR analysis using a High Fidelity One-Step RT-PCR Kit (Takara, Dalian, China). The forward/reverse primers, F1/R1 (Table 1), were designed for amplifying a 530-bp fragment. The RT-PCR reaction underwent 50 °C for 30 min, 94 °C for 2 min and then 30 cycles at 98 °C (10 s), 55 °C (5 s), and 72 °C (35 s). The plasmids p15A-SVA-p16, cDNA of wild-type SVA (wt-SVA-cDNA) and ddH2O were simultaneously subjected to PCR analysis as control groups using the F1/R1 primer pair. The PCR reaction contained 2 × PrimeSTAR Max Premix (Takara, Dalian, China) and underwent 30 cycles at 98 °C (10 s), 55 °C (5 s), and 72 °C (10 s). RT-PCR and PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing for analyzing the RT-PCR product. SVA-iLOV only needs to be detected by fluorescence observation.

The culture supernatant of rSVA-eGFP at passage 5 (P5) was harvested for extracting viral RNA by a Viral RNA/DNA Extraction Kit (Takara, Dalian, China). The extracted RNA was used as template for RT-PCR analysis using a High Fidelity One Step RT-PCR Kit (Takara, Dalian, China). The forward/reverse primers, F1/R1 (Table 1 and Figure 1A), were designed for amplifying a 693-bp fragment. The RT-PCR reaction underwent 45 °C for 10 min, 94 °C for 2 min and then 30 cycles at 98 °C (10 s), 55 °C (15 s) and 68 °C (10 s). The extracted RNA was simultaneously subjected to PCR analysis as a control using the F1/R1 primer pair. The PCR reaction contained 2 × PrimeSTAR Max Premix (Takara, Dalian, China) and underwent 30 cycles at 98 °C (10 s), 55 °C (5 s) and 72 °C (10 s). RT-PCR and PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing for analyzing the RT-PCR product.