Infusion 2.0 dry down pcr cloning kit

The 3′ untranslated region (UTR) of the POLD1 luciferase reporter plasmid (pHS-AVC-LW2301) was obtained from SyngenTech (Beijing, China). For the assessment of CTCF function on POLD1 promoter activity, genomic fragments harboring putative CTCF-binding sites in the human POLD1 promoter (∼1.8 kb upstream of the translation start site) were subcloned into the pGL4-Luc reporter vector (Promega) using Infusion 2.0 Dry-Down PCR cloning kit (Clontech, Shanghai, China). Promoter activity was further validated by mutation of the putative CTCF-binding site on the promoter at −1,015 to −997 or −625 to −607 by selecting these sites. For the luciferase reporter assay, 293T cells were seeded in a 24-well plate and incubated for 24 h before transfection. Subsequently, luciferase constructs, POLD1, and POLD1 + CTCF were co-transfected into 293T cells using Lipofectamine 3000. Cells were collected at 48 h after transfection and measured using the Dual-Luciferase Reporter System (Promega, WI, United States), according to manufacturer’s protocols. Three independent experiments were performed, and data were presented as mean ± SD.

The genomic fragments harboring the putative miR-34a-binding sites in human OAZ2 3′UTR were subcloned into the pGL3-Luc reporter vector (Promega, Beijing, China) using Infusion 2.0 Dry-Down PCR cloning kit (Clontech, Shanghai, China). Promoter activity was further validated by mutation of the putative miR-34a-binding site on the OAZ2 3′UTR by replacing ACAC with CACA using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA). For reporter assay, 0.5 μg of pGL3-OAZ2 WT or pGL3-OAZ2 Mu, along with 0.05 μg of pRL-SV40 plasmid and miR-34a mimic, was cotransfected into HeLa cells using FuGENE®6 (Promega). 48 h later, cells were harvested and subjected to luciferase activity measurements using the Promega Dual-Luciferase® Reporter Assay System.