Inserts for HOXA11-AS and IBSP luciferase vectors were amplified with the PC3 genome as template, using the primers listed in Table S1 . The insert of HOXA11-AS luciferase vector containing the promoter region ranged from −1468 to +383. The insert of HOXA11-AS luciferase vector without the promoter region ranged from −367 to +383. Both inserts were cloned into pGL3-Basic luciferase vector (Promega, Madison, WI, USA) using KpnI and HindIII restriction enzyme sites. The promoter region of the IBSP luciferase vector ranged from −344 to +90. An insert of 269 bp within this region was cloned into the pGL3-Basic luciferase vector (Promega) with KpnI and SacI restriction enzyme sites, using primers listed in Table S1 .
Pgl3 basic luciferase vector
Manufactured by Promega
Sourced in United States
The PGL3-Basic luciferase vector is a tool used in molecular biology and genetic research. It contains a firefly luciferase gene that can be used to measure gene expression levels in cells. The vector provides a basic backbone for cloning and expressing target genes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (34 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (18 mentions)
Dual luciferase reporter assay kit, by Promega (9 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (8 mentions)
Dual luciferase assay system, by Promega (8 mentions)
Dual luciferase reporter system, by Promega (5 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Prl tk vector, by Promega (4 mentions)
Glomax 96 luminometer, by Promega (4 mentions)
Bright glo luciferase assay system, by Promega (3 mentions)
Prl tk plasmid, by Promega (3 mentions)
Luciferase reporter assay kit, by Promega (3 mentions)
Pgpu6 gfp neo vector, by GenePharma (2 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (2 mentions)
Enspire multimode plate reader, by PerkinElmer (2 mentions)
Prl tk, by Promega (2 mentions)
Pgl3 her2 luc, by Promega (2 mentions)
Pgl3 her3 luc, by Promega (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
138 protocols using pgl3 basic luciferase vector
1
Cloning Luciferase Vectors for HOXA11-AS and IBSP
2
Constructing V5-tagged human SM22 and mutants
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pCMV6-XL5-hSM22 was purchased from Origene Inc (#sc118118). To construct the C-terminal V5 tagged human SM22 and its truncation mutants, an EcoRI-XhoI-BamHI-KpnI-V5-XbaI fragment was subcloned into EcoRI and XbaI sites of pcDNA3.1. The resulting construct was cleaved with HindIII and XhoI followed by insertion of the PCR fragments containing the full length V5-tagged human SM22 or its truncation mutants. Clones were verified by sequencing (Genewiz Technologies, INC).
The pCGN-SRF plasmid and the luciferase reporter driven by the 4xCArG boxes from the fos promoter were generous gift from Dr. Eric Olson [28 (link), 29 (link)]. The 1.4kb mouse Egr3 promoter (upstream of the ATG) containing the CArG box (CCATATATGG) was PCR cloned into the XhoI/HindIII sites of pGL3-basic luciferase vector (Promega). About 2kb rat Nik promoter (upstream of the ATG) containing the CArG box (CCAACAATGG) was PCR cloned into the NheI/HIII sites of pGL3-basic luciferase vector (Promega). The CArG box mutant (TTAACAATT) was subsequently generated as the Nik2kbCArGmut-luc. All constructed luciferase reporters containing the promoter fragment or the CArG box mutant were verified by sequencing (Genewiz Technologies, INC). All plasmids DNA were prepared for transfection using the plasmid Prep kit (Qiagen).
The pCGN-SRF plasmid and the luciferase reporter driven by the 4xCArG boxes from the fos promoter were generous gift from Dr. Eric Olson [28 (link), 29 (link)]. The 1.4kb mouse Egr3 promoter (upstream of the ATG) containing the CArG box (CCATATATGG) was PCR cloned into the XhoI/HindIII sites of pGL3-basic luciferase vector (Promega). About 2kb rat Nik promoter (upstream of the ATG) containing the CArG box (CCAACAATGG) was PCR cloned into the NheI/HIII sites of pGL3-basic luciferase vector (Promega). The CArG box mutant (TTAACAATT) was subsequently generated as the Nik2kbCArGmut-luc. All constructed luciferase reporters containing the promoter fragment or the CArG box mutant were verified by sequencing (Genewiz Technologies, INC). All plasmids DNA were prepared for transfection using the plasmid Prep kit (Qiagen).
3
Luciferase Assays for lncEGFL7OS/EGFL7 Promoter
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Luciferase assays were performed as described (Wang et al., 2008a (link)). The putative bidirectional promoter for lncEGFL7OS/EGFL7 was PCR amplified from human DNA and cloned into promoterless PGL3 Basic luciferase vector (Promega). Primers include: plncEGFL7OSup (XhoI): 5’-atcgCTCAGATAGACTCTGATGGCCCAGG -3’ and plncEGFL7OSdn (XhoI): 5’ –atcgCTCAGACCAGCTTGGTGCAGGGAG -3’. 293 T cells in 24-well plates were transfected with 50 ng of reporter plasmids in the presence or absence of increasing amount of Ets1 or Ets1 DNA-binding mutant expression plasmid.
4
NDRG1 Promoter Cloning and Truncation
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The NDRG1 promoter region (−800~ + 515 bp) was amplified from human genomic DNA. The primers with MluI and HindIII restriction sites are listed in Table 1 . The PCR product was cloned into the pJET1.2/blunt cloning vector using the CloneJET PCR cloning kit (Fermentas, Thermo Scientific, Waltham, MA, USA). The NDRG1 promoter fragment was then digested with MluI and HindIII (TaKaRa, Shiga, Japan), and subcloned into the pGL3 Basic luciferase vector (Promega, Madison, WI, USA). Serial 5′-deleted NDRG1 promoters prepared by PCR amplification using forward primers containing MluI restriction sites and reverse primers containing HindIII restriction site are listed in Table 1 . The truncated NDRG1 promoter fragments were all subcloned into the pGL3 Basic luciferase vector at the MluI/HindIII sites.
5
Cloning and Luciferase Assay of CD47 Regulatory Regions
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The fragment of the CD47 regulatory regions was cloned by PCR using human genomic DNA as a template. The fragment was then cloned into the luciferase reporter plasmid pGL3-Basic Luciferase Vector (Promega). Cells were transiently transfected with desired pGL3 basic-based constructs. Luciferase activity was measured using the Dual-Glo® Luciferase Assay kit (Promega) with a Synergy 2 multi-detection microplate reader (BioTek, VT).
6
Cloning and Modulation of SPOP in FHC Cells
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The full-length ORF of SPOP (1125 bp, NM_001007226.1) was amplified from cDNA of FHC cells. The primers were as follows: F: 5′-AGAGAATTCATGTCAAGGGAAATCTTTGC-3′, R: 5′-AGAGGATCCTTAGGATTGCTTCAGGCGTT-3′. To construct the lentivirus production containing SPOP, the ORF of SPOP was subcloned into the pLenti-CMV-GFP vector (Addgene, Cambridge, MA, USA).
The synthesized DNA fragments encoding the short-hairpin RNA (shRNA) used for the knockdown of endogenous SPOP were inserted into the pGPU6/GFP/Neo vector (GenePharma, Shanghai, China). The sequences of the shRNAs were as follows: shSPOP, 5′-AACGCCTGAAGCAATCCTACTCGAGTAGGATTGCTTCAGGCGTT-3′, NC, 5′-TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′. All plasmids were verified by sequencing.
Serial deletion fragments of SPOP promoter region (−1320, −1167, −655, −350, −221, −189, −10/+472) were amplified by PCR and cloned into a pGL3-basic luciferase vector (Promega). A NheI site was added to the 5′-primer, and a XhoI site was added to the 3′-primer. The constructs were verified by sequencing of both DNA strands. The sequences of mutated sites were synthesized by Genescript (Nanjing, China).
FLAG-Gli2, HA-Ub and pcDNA3.1(-)A-RXRA were purchased from GenePharma.
The synthesized DNA fragments encoding the short-hairpin RNA (shRNA) used for the knockdown of endogenous SPOP were inserted into the pGPU6/GFP/Neo vector (GenePharma, Shanghai, China). The sequences of the shRNAs were as follows: shSPOP, 5′-AACGCCTGAAGCAATCCTACTCGAGTAGGATTGCTTCAGGCGTT-3′, NC, 5′-TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′. All plasmids were verified by sequencing.
Serial deletion fragments of SPOP promoter region (−1320, −1167, −655, −350, −221, −189, −10/+472) were amplified by PCR and cloned into a pGL3-basic luciferase vector (Promega). A NheI site was added to the 5′-primer, and a XhoI site was added to the 3′-primer. The constructs were verified by sequencing of both DNA strands. The sequences of mutated sites were synthesized by Genescript (Nanjing, China).
FLAG-Gli2, HA-Ub and pcDNA3.1(-)A-RXRA were purchased from GenePharma.
7
Transcriptional Regulation of Murine Ccl2 Promoter
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The promoter sequences for murine Ccl2 genes were obtained from the National Center for Biotechnology Information database. Murine Ccl2 promoter fragments were amplified by PCR of murine genomic DNA using the following S and AS primers: 5′-TCCGGCCCATGAGAGAACTGCTT-3′ (S1, –2468/–2445), 5′-ACTATGCCTGGCTCCTGGTA-3′ (S2, –1401/–1381), 5′-GAAGACTCCGCTCAGCCAC-3′ (S3, –1136/–1117), and 5′-TGGCTTCAGTGAGAGTTGGCTGGT-3′ (AS, +67/+44). The Ccl2 promoter nucleotide sequence was confirmed by DNA sequencing and cloned into a pGL3 basic luciferase vector (Promega) to generate a reporter plasmid. We transfected BNL-CL2 with either cytomegalovirus promoter-T7–tagged FoxM1 plasmid (CMV-T7-FoxM1) or cytomegalovirus-empty expression plasmid, as well as with luciferase reporters driven by the murine Ccl2 promoter lesions. After seeding cells and culturing them overnight, the plasmid was transfected with FuGENE HD transfection reagent (catalog E2311; Promega) according to the manufacturer’s instructions. We used the 6×CDX2-Luc plasmid, a known FoxM1 reporter, as a positive control for CMV-T7-FoxM1 transcriptional activity. The CMV-Renilla luciferase plasmid was used as internal controls to normalize transfection efficiency. A dual luciferase assay (Promega) was performed 48 hours after transfection, as described previously.38 (link)
8
Evaluating LncPGCR Promoter Activity
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The amplified promoter of LncPGCR and different deletion fragments of the LncPGCR promoter were subcloned into the firefly plasmids in the pGL3Basic luciferase vector (Promega). Different length PCR amplification primers of the LncPGCR promoter region are shown in Supplementary Table S1 . TCF7L2 binding site mutation primers are shown in Supplementary Table S2 . Finally, a Dual-Luciferase Reporter Assay System (Promega) was used to evaluate luciferase activity. Each procedure of these experiments was repeated independently three times.
9
Luciferase Assay for NLRP3 Regulation
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Wild-type and mutant NLRP3 were constructed into pGL3-Basic luciferase vector (Promega, Madison, WI, USA) to produce luciferase reporter plasmids. HCAECs were transfected with the plasmids and then were cotransfected with miR-NC or miR-30b-5p mimic with Lipofectamine 2000 (Life Technologies, New York, USA). The luciferase activity was determined by Promega Kit (Promega) after cells were transfected for 48 h.
10
Dual-Luciferase Assay for miR-942-5p Targets
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Dual-luciferase reporter assays were performed to verify further whether hsa_circ_0002062 or CDK6 was the target of hsa-miR-942-5P.
hsa_circ_0002062 wild type, hsa_circ_0002062 mutant, CDK6 wild type, or CDK6 mutant inserts were inserted into the pGL3-basic luciferase vector (Promega, Madison, WI, United States) in order to generate report plasmids. Afterward, cells transfected with indicated reporter plasmid were cultured in a 6-well plate and co-transfected with 5-μL miR-NC, or hsa-miR-942-5p mimic using Lipofectamine 2000 (Invitrogen). The Dual-Luciferase Reporter Gene Assay System (E1910, Promega, United States) was used to detect luciferase activity according to the manufacturer’s instruction, and the ratio of firefly to renilla luciferase activity was calculated.
hsa_circ_0002062 wild type, hsa_circ_0002062 mutant, CDK6 wild type, or CDK6 mutant inserts were inserted into the pGL3-basic luciferase vector (Promega, Madison, WI, United States) in order to generate report plasmids. Afterward, cells transfected with indicated reporter plasmid were cultured in a 6-well plate and co-transfected with 5-μL miR-NC, or hsa-miR-942-5p mimic using Lipofectamine 2000 (Invitrogen). The Dual-Luciferase Reporter Gene Assay System (E1910, Promega, United States) was used to detect luciferase activity according to the manufacturer’s instruction, and the ratio of firefly to renilla luciferase activity was calculated.
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