Mutant A3B DNA Amplification and Cloning

HEK293T cells were transfected with pDON-EGFP, pEF-UGI, and expression vectors for A3B WT or its mutants, using the XtremeGENE HP DNA Transfection Reagent. After two-day cultures, total DNA was extracted using the Quick Gene DNA whole blood kit (KURABO). First round PCR was performed using the primers and rTaq DNA polymerase (Takara), with the following reaction profile: 30 s at 94 °C, 25 cycles of 30 s at 94 °C, 40 s at 62 °C, and 90 s at 72 °C, followed by 10 min at 72 °C^{20 (link)}. The amplicons were separated by electrophoresis in 1% (w/v) agarose gel, and extracted with the FastGene Gel/PCR Extraction kit (NIPPON Genetics). We used 100 ng of first-round PCR products as templates for nested PCR using KOD Fx Neo (TOYOBO), with the following reaction profile: 5 min at 95 °C, 30 cycles of 10 s at 81–88 °C, 30 s at 62 °C, and 60 s at 72 °C, followed by 5 min at 72 °C. The amplicons derived at each of the indicated denaturation temperatures were then cloned into the pT7-blue vector (Novagen).