Publication protocol
The first round of gene amplification was carried out for 5 cycles (95°C for 1 minute, 65°C for 50 seconds, 72°C for 45 seconds), followed by the addition of 0.8 μL of 10 pmol primers (forward 1 and reverse 2 primers, Table 1). The second round of amplification was performed for 28 cycles (95°C for 40 seconds, 60°C for 50 seconds, 72°C for 45 seconds), and a final extension of 72°C for 3 minutes. The final PCR products (1645 bp) were purified using a commercial kit (Bioneer, South Korea) according to the manufacturer’s recommendations. The amplified igVR-1a chimera gene and JFH1 vector were digested using the KpnI and BsiWI restriction enzymes (New England Biolabs, UK) and the digested products were purified using a commercial kit (Bioneer, Korea) according to the manufacturer’s recommendations. The ligation mixture was prepared as follows: 0.8 µL of T4 DNA ligase (Fermentas, USA), 1 µL of 10× T4 DNA ligase buffer, 180 ng of purified linear JFH1 vector, 50 ng of purified igVR-1a chimera fragment, and 0.5 µl of 50% PEG 4000 solution. This mixture was prepared and incubated at 14°C overnight. The ligation product was transformed into E. coli JM109 strain competent cells, as described previously (12). The presence of the igVR-1a chimera clone was confirmed by colony PCR using the forward 1 and reverse 2 specific primers (Table 1) and a restriction enzyme analysis. The confirmed cloned fragments were used for purification with a commercial kit (Qiagen, USA) according to the manufacturer’s recommendations.
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