14 oligonucleotide gBlocks (IDT), ranging in 500–1000 nt in length, and corresponding to 10 enhancer regions were synthesized. Each gBlock contained a constant 5′ GCTAGCCTCGAGGAT and 3′ ATCAAGATCTGGCCT region, for direct cloning into an EcoRV (NEB) linearized minimal promoter firefly luciferase vector pGL4.23[luc2/minP] (Promega). The resulting reporter constructs were verified by DNA sequencing. BV-2 cells were kindly provided by Dr. Bruce Yankner. N2a cells were purchased from the American Type Culture Collection and maintained following their protocols. Briefly, cells were grown in RPMI-1640 and DMEM respectively, supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep, and split 1:10 every 3 days. Cells were seeded into 24-well plates 1 day before transfection. Transfections into BV-2 and N2a cells were performed with 1 μg of a pGL4.23 plasmid and 200 ng of Renilla luciferase construct pGL4.74[Rluc/TK] (Promega). Luciferase activities were measured 24 h post-transfection using the Dual-Glo Luciferase Assay (Promega) and an EnVision 2103 Multilabel Plate Reader (PerkinElmer) and normalized to Renilla luciferase activity. All assays were performed in triplicate.
Pgl4.74 rluc tk
Manufactured by Promega
The PGL4.74[Rluc/TK] is a plasmid vector that contains a Renilla luciferase (Rluc) reporter gene and a Herpes simplex virus thymidine kinase (TK) gene. It is designed for use in various experimental applications.
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2 protocols using pgl4.74 rluc tk
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Enhancer Reporter Construct Synthesis
2
Enhancer Reporter Construct Synthesis
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14 oligonucleotide gBlocks (IDT), ranging in 500–1000 nt in length, and corresponding to 10 enhancer regions were synthesized. Each gBlock contained a constant 5′ GCTAGCCTCGAGGAT and 3′ ATCAAGATCTGGCCT region, for direct cloning into an EcoRV (NEB) linearized minimal promoter firefly luciferase vector pGL4.23[luc2/minP] (Promega). The resulting reporter constructs were verified by DNA sequencing. BV-2 cells were kindly provided by Dr. Bruce Yankner. N2a cells were purchased from the American Type Culture Collection and maintained following their protocols. Briefly, cells were grown in RPMI-1640 and DMEM respectively, supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep, and split 1:10 every 3 days. Cells were seeded into 24-well plates 1 day before transfection. Transfections into BV-2 and N2a cells were performed with 1 μg of a pGL4.23 plasmid and 200 ng of Renilla luciferase construct pGL4.74[Rluc/TK] (Promega). Luciferase activities were measured 24 h post-transfection using the Dual-Glo Luciferase Assay (Promega) and an EnVision 2103 Multilabel Plate Reader (PerkinElmer) and normalized to Renilla luciferase activity. All assays were performed in triplicate.
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