A −400 bp Mymsl promoter luciferase reporter was PCR amplified from mouse genomic DNA and cloned into the pGL3 basic luciferase reporter (Promega). The mutation for the predicted CArG box was performed using QuikChange II Site-Directed Mutagenesis Kit (Cat. #200523, Agilent). Primers for the PCR amplification and mutagenesis are included in Supplementary Table 1 . Mutation of the CArG element was validated by Sanger sequencing at Cornell University Life Sciences Core Laboratories Center. Luciferase assay was carried out as previously described [17 (link)]. Briefly, 10T1/2 cells were plated in 24-well cell culture plates until 80% confluency and then co-transfected with luciferase reporter plasmids, ± SRF or MYOCD expression plasmids, and Renilla as an internal control. Luciferase activity was examined at 36 hrs post-transfection using a Dual Luciferase Assay Kit per the vendor’s protocol (Promega).
Pgl3 basic luciferase reporter
Manufactured by Promega
Sourced in United States
The PGL3 basic luciferase reporter is a plasmid vector that contains the firefly luciferase gene as a reporter. This vector can be used to measure gene expression in a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Pcr xl topo, by Thermo Fisher Scientific (2 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Pmir report vector, by Thermo Fisher Scientific (2 mentions)
Mlu1 1, by Takara Bio (2 mentions)
Xhoi, by Takara Bio (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Pgfp c3, by Takara Bio (1 mentions)
Pnf κb luc and control plasmids, by Takara Bio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
Pcdna3.0 expression vector, by Thermo Fisher Scientific (1 mentions)
Sirnas, by RiboBio (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Plxsn, by Takara Bio (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Dual luciferase reporter gene assay kit, by Beyotime (1 mentions)
Pgl3 promoter vector, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using pgl3 basic luciferase reporter
1
Mymsl Promoter Luciferase Assay
2
TNIP2 3'UTR Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TNIP2 3ʹ-UTR was PCR amplified from NBEC RNA and cloned into the SacI/XmaI restriction sites of the pGL3-basic luciferase reporter (Promega, Madison, WI, USA) and pGFP-C3 (Clontech, Mountain View, CA, USA) plasmids. The miR-423 mimics, negative control, and anti-miR-423 inhibitor were purchased from RiboBio (Guangzhou, Guangdong, China). pNF-κB-luc and control plasmids (Clontech, Mountain View, CA, USA) were used to quantitatively examine NF-κB activity. Transfection of plasmids was performed using the Lipofectamine 2000 reagent (Invitrogen).
3
Cloning Grin2A promoter for Luciferase assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two kilobases of the Grin2A promoter (5′ from the ATG) was amplified with high-fidelity Phusion DNA polymerase from the genomic DNA of WT C57B/6 mice. The following primers were used: F-5′-GGTACCAGCTCCTGGTCGCACAA and R-5′-CTCGAGTAGGGTCCCTGTAAGGTGGA. These primers also introduced KpnI and XhoI restriction sites at the 5′ and 3′ ends, respectively. The amplicon was digested with KpnI and XhoI and directionally cloned into a pGL3 basic luciferase reporter (Promega). pGL3:Grin2A clones were screened by double digest and verified by DNA sequencing. Empty basic pGL3 plasmids were used as negative controls, and the luciferase driven by cytomegalovirus (CMV) promoter was used as a positive control.
4
Construction of pMXs-Flag-Dlx3-IRES-EGFP and Dlx Vectors
5
Transcriptional Regulation of DIO3OS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The promotor region of DIO3OS containing wild type (wt) or mutant type (mut) of predicted Smad2/Smad3/Smad4 complex recognition site was constructed into a PGL3-Basic luciferase reporter (Promega, USA). The luciferase reporters were co-transfected into cells together with expression plasmids of Smads (Smad2/Smad3/Smad4). a Dual-Luciferase reporter assay system (Promega) was employed to test luciferase activities after 48 h.
The wild type or mutant type fragment of DIO3OS, CTGF 3’UTR or ZEB1 3’UTR containing predicted miR-656-3p or miR-485-5p binding site was constructed into a pMIR-REPORT vector (Thermo Scientific). The luciferase reporters were co-transfected into cells together with miRNAs mimics. Luciferase reporter assay system (Promega) was employed to test luciferase activities after 48 h.
The wild type or mutant type fragment of DIO3OS, CTGF 3’UTR or ZEB1 3’UTR containing predicted miR-656-3p or miR-485-5p binding site was constructed into a pMIR-REPORT vector (Thermo Scientific). The luciferase reporters were co-transfected into cells together with miRNAs mimics. Luciferase reporter assay system (Promega) was employed to test luciferase activities after 48 h.
6
Cloning and Luciferase Reporting of LINC01189 and ZEB1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full-length cDNAs of human LINC01189 and ZEB1 were synthesized and cloned into pcDNA3.0 expression vector (Life Technologies). The miR-586 mimic/inhibitor, siRNAs, and the corresponding controls were purchased from RiboBio (China) and are listed in Table S1 . For the promoter luciferase reporter assay, the promoter regions of LINC01189 (−2,500 to +1) or ZEB1 (−1,000 to +1) and the promoter region with mutated ZEB1 or TCF4 binding site were synthesized and cloned into pGL3-basic luciferase reporter (Promega). For miRNA target gene luciferase reporter assays, target sequences containing predicted miRNA binding sites or corresponding mutants were synthesized and cloned into the psiCHEK2 luciferase vector.
7
Plasmid Construction for Dlx3 and AhR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To construct pMXs-Flag-Dlx3-IRES-EGFP, an EcoRI/XhoI fragment generated by PCR amplification of mouse and human Dlx3 from the PCR cloning vector PCR-XL-Topo (Invitrogen, Carlsbad, CA, USA) was sub-cloned into the EcoRI/XhoI site of pMXs-Flag-IRES-EGFP (a kind gift from Wayne Yokoyama, Washington University, St. Louis, MO, USA). pMXs-Flag-mDlx1 and -mDlx2 were also constructed by sub-cloning PCR products. pEGFP-Oct4 was constructed by sub-cloning Oct4 PCR products into pEGFP-C1 (Clontech Laboratories, Mountain View, CA, USA). Promoter regions AhR-8 (−5638/−5403), AhR-8/9 (−5638/−3981) and AhR-P (−1805/−375) of the AhR gene were incorporated into the XhoI/HindIII sites of the pGL3 basic luciferase reporter (Promega, Sunnyvale, CA, USA) using PCR amplification and sub-cloning to generate pGL3-basic-AhR vectors.
8
Viral and cellular gene expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular and viral genes were expressed using the retroviral vector pLXSN (Clontech, Palo Alto, CA) or the expression vector pcDNA-3 (Invitrogen). The pLXSN-LMP-1 and the mutants LMP-1AxAxA, LMP-1 378 stop, and LMP-1AxAxA/378 stop constructs have been previously described [41] . The pGL3 basic luciferase reporter (Promega) and pGL3 containing the DOK1 promoter constructs have been described previously [29] (link), The NF-κB super-repressor Δ-IκBα, which lacks the coding sequence of the first 36 N-terminal amino-acids, was kindly provided by Dr Elliot Kieff (Harvard Medical School, Boston, Massachusetts, USA). The expression plasmids pDEST-myc-EBNA1, pSG5-EBNA2, pDEST-myc-EBN3A1, pDEST-myc-EBNA3B, pDEST-myc-EBNA3C were kindly provided by Dr Evelyne Manet (ENS, Lyon, France).
9
HOIL-1L Promoter-Driven Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HOIL-1L promoter region (2.0 kb upstream of transcription start site) was cloned into the vector pGL3-Basic-luciferase reporter (Promega) to get pGL3-HOIL-1L-P. 293T cells were seeded on 24-well plates and co-transfected with the Renilla luciferase reporter pSV-40 and pGL3-HOIL-1L-P or pGL3-Basic. 48 h later the luciferase activity was measured by dual-luciferase reporter assay according to the instruction, the HOIL-1L promoter luciferase was normalized by Renilla luciferase activity.
10
Generation of MMP-1 Promoter Reporter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate a reporter construct of the human MMP-1 promoter (GenBank Accession No. NM_000011.10), a 1938-bp DNA fragment including the promoter of the human MMP-1 gene (−1880 to +40) was PCR-amplified using genomic DNA from human dermal fibroblasts as a template, PrimeSTAR® GXL DNA polymerase (TaKaRa, Shiga-ken, Japan), and the primer pair 5’-GAAGCTAGCTCCCTCACAGTCGAGTATATCTGCCAC-3’, which includes a NheI site (italicized) and 5’-GAAAAGCTTGCAAGGTAAGTGATGGCTTCCCAG-3’, which includes a HindIII site (italicized). The PCR product cleaved with NheI and HindIII was cloned into the pGL3-Basic luciferase reporter (Promega, Madison, WI, USA) that was digested with NheI and HindIII to generate the pGL3-MMP-1 promoter.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!