Kod plus 2 dna polymerase

In a 150-mm dish, 1 x 10⁷ of Huh7.5 cells were seeded 1 day before transduction and infected with 5 x 10⁶ infectious dose of the IFN cDNA carrying lentiviral vectors for 24 h. After 48 h post-transduction, the cells were challenged with DENV-2 (EDEN2 3295) at an MOI of 1. The culture medium was changed every 2–3 days, and after 2 weeks, cell colonies that survived the DENV challenge were transferred to 48-well plates and expanded for further analysis.
Genomic DNA was isolated from the resistant clones using the Wizard Genomic DNA Purification Kit (Promega) from cells that displayed low infectivity of DENV in immunofluorescence and plaque assay. The cDNA was then amplified by PCR using KOD-Plus 2 DNA polymerase (Toyobo) and primers (5’-CTT CCA TTT CAG GTG TCG TGA ACA CGC TAC CGG TCT CGA G-3’ and 5’-CAA ACG CAC ACC GGC CTT ATT CCA AGC GGC TTC GGC CAG-3’) flanking the Gateway cassette in the pYK005c lentiviral vector. cDNA was further amplified by nested PCR using primers (5’-ACC GGT CTC GAG AAT TAT CAA CAA-3’ and 5’-GCT GCA GAA TTA TCA ACC ACT TTG-3’) and cloned into the pCR-Blunt II-TOPO vector (Life Technologies). The sequence of cDNA in the pCR-Blunt II-TOPO vector was analyzed by an automated DNA sequencer, and the data was compared with the DNA database at the National Center for Biotechnology Information using a BLAST search.

E. coli SCS110 was used to avoid DNA methylation, and polymerase chain reaction (PCR) was conducted using KOD-Plus2 DNA polymerase (Toyobo, Osaka, Japan). Plasmid DNA was purified using a LaboPass™ Plasmid Mini Purification Kit (Cosmo Bio Co., Ltd., Tokyo, Japan).