Luciferase reporter constructs were used, and luciferase assays were performed as described previously. Briefly, the mouse Nrdp1 3′-UTR sequence was amplified by PCR from mouse genomic DNA and ligated into the pMIR-REPORT luciferase vector multiple cloning site (Ambion, Austin, TX) to yield pMIR-Nrdp1 3′-UTR (NRDP1 3′-UTR). Another pMIR-REPORT luciferase construct containing the Nrdp1 mRNA 3′-UTR with a mutation by site-directed mutagenesis was generated as a negative control and named Mut-Nrdp1 3′-UTR. Macrophages were plated in 6-well plates and allowed to reach 60-80% confluence overnight. Cells were then co-transfected with a reporter construct (pMIR-null REPORT plasmid, pMIR- Nrdp1 3′-UTR, pMIR-Nrdp1 3′-UTR-Mut). After 24 h, cells were harvested, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s recommendations. Luciferase activity was normalized to control TK Renilla construct expression (pRL-TK, Promega).
Pmir report luciferase vector multiple cloning site
Manufactured by Thermo Fisher Scientific
The PMIR-REPORT luciferase vector multiple cloning site is a DNA vector designed for the expression and measurement of luciferase reporter genes. It contains a multiple cloning site where target DNA sequences can be inserted for gene expression studies.
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Lab products found in correlation
3 protocols using pmir report luciferase vector multiple cloning site
1
Luciferase Assay for Nrdp1 3'UTR Regulation
2
Luciferase Assay for Nrdp1 3'-UTR Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Luciferase reporter constructs were used, and luciferase assays were performed as described previously. Brie y, the mouse Nrdp1 3′-UTR sequence was ampli ed by PCR from mouse genomic DNA, and ligated into the pMIR-REPORT luciferase vector multiple cloning site (Ambion, Austin, TX) to yield pMIR-Nrdp1 3′-UTR (NRDP1 3′-UTR). Another pMIR-REPORT luciferase construct containing the Nrdp1 mRNA 3′-UTR with a mutation by site-directed mutagenesis was generated as a negative control and named Mut-Nrdp1 3′-UTR. Macrophage were plated in 6-well plates and allowed to reach 60%-80% con uence overnight. Cells were then co-transfected with a reporter construct (pMIR-null REPORT plasmid, pMIR-Nrdp1 3′-UTR, pMIR-Nrdp1 3′-UTR-Mut). After 24 h, cells were harvested, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's recommendations.
Luciferase activity was normalized to control TK Renilla construct expression (pRL-TK, Promega).
Luciferase activity was normalized to control TK Renilla construct expression (pRL-TK, Promega).
3
Luciferase Assay for Nrdp1 3'-UTR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Luciferase reporter constructs were used, and luciferase assays were performed as described previously. Briefly, the mouse Nrdp1 3′-UTR sequence was amplified by PCR from mouse genomic DNA, and ligated into the pMIR-REPORT luciferase vector multiple cloning site (Ambion, Austin, TX) to yield pMIR-Nrdp1 3′-UTR (NRDP1 3′-UTR). Another pMIR-REPORT luciferase construct containing the Nrdp1 mRNA 3′-UTR with a mutation by site-directed mutagenesis was generated as a negative control and named Mut-Nrdp1 3′-UTR. Macrophage were plated in 6-well plates and allowed to reach 60%-80% confluence overnight. Cells were then co-transfected with a reporter construct (pMIR-null REPORT plasmid, pMIR-Nrdp1 3′-UTR, pMIR-Nrdp1 3′-UTR-Mut). After 24 h, cells were harvested, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's recommendations. Luciferase activity was normalized to control TK Renilla construct expression (pRL-TK, Promega).
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