Qiaamp dna mini and blood mini kit

Genomic DNA was extracted from cultured NHAs using a QIAampDNA mini and blood mini kit (Qiagen, Hilden, Germany). A 1.3kb promoter region of IL-8 was amplified using the primers KR9 (F-5’-ctggcctaactggccggtacGGTGTCCTTGGATAAAGAG-3’) and KR10 (R-5’-gaggccagatcttgatatccTACCAAAGCATCAAGAATAG-3’) and inserted into KpnI and XhoI linearized pGL4.12 vector via Gibson Assembly (New England Biolabs, Ipswich, MA). The following PCR conditions were used: initial denaturation at 95 °C for 10 min; followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 60 °C for 30 sec, and extension at 72 °C for 1.5 min and a final extension at 72 °C for 10 min using the Q5 hot start Taq polymerase. Recombinant plasmid was subjected to restriction digestion with SfiI and sequencing to confirm the presence of a single copy of insert in proper orientation. This wild type IL-8 plasmid (p576) was subjected to site-directed mutagenesis using the Q5 site-directed mutagenesis kit (New England Biolabs) with primers, (F-5’-TTTAAAGATCctgGAAAACTTTCGTCATACTC-3’ and R-5’-ATAATTTAATTTTAATATACATTTAAAATACTG-3’) to obtain the substitution plasmid at the TCF/LEF binding site (p594). This plasmid was sequenced from both directions to confirm proper substitution.