Icycler thermal cycler pcr system

Extraction of viral DNA was performed with the High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany), from the Vero-adapted Ba71V isolate. DNA fragments corresponding to A104R ORF were amplified by PCR using the following primers: A104R_Fw GGA CTA GTA TGT CGA CAA AAA AAA AGC CCA CAA TTA and A104R_Rev TCC CCC GGG TTA ATT TAA CAT ATC ATG AAC AGG TTT CAA TGC. A proof reading polymerase (Phusion^® High-Fidelity DNA Polymerase, Thermo Scientific) was used according to the manufacturer’s instructions (50 µL of master mix: 1 µL of forward and reverse primers (0.5 µM each), 10 µL of Phusion HF buffer, 1 µL dNTPs (200 µM), 0.5 µL Phusion DNA polymerase, 24.5 µL of Milli-Q water and 2 µL of DNA). All PCR reactions were performed in the iCycler Thermal Cycler PCR system (Bio-Rad, Munich, Germany), with the following thermal profile: initial denaturation at 98 °C for 1 min followed by 35 cycles of 10 s at 98 °C, 60–72 °C for 30 s and 72 °C for 30 s per kb), and final extension at 72 °C for 5 min. Following the PCR amplification, the obtained DNA fragments were cloned into different vector depending of the subsequent protocol. For practical reasons, each set of primers used to amplify the DNA fragments corresponding to the homology arms of A104R ORF and bacterial GUS gene (selection marker) are described in the following sections.

Total RNA was isolated from liver or epididymis white fat tissue grinding fluid using Trizol reagent (TIANGEN, China). Complementary DNA was synthesized using a Quantscript RT Kit (TIANGEN, China) and the products were used as template for RT-PCR. The primers used are shown in the Supplementary Materials Table S2. Real-time PCR was performed on an iCycler Thermal Cycler PCR System (Bio-Rad Laboratories, Hercules, USA). The following general RT-PCR protocol was used: denaturation program (95 °C for 5 min); a three-segment amplification program repeated 40 times (95 °C for 30 s, annealing temperature for 45 s and 72 °C for 45 s); and extension (72 °C for 10 min). All samples were conducted in triplicate. Data acquisition and subsequent data analyses were performed using Quantity One 1-D Analysis Software (Bio-Rad).