The promoter region of the CXCR4 and CXCR7 genes, from -2,000 bp to +50 bp, relative to the transcription start site were amplified according to previous report [24 (link)]. The confirmed sequence was then inserted into a pGL3-Basic vector (Promega). The pGL3-Basic vector was digested by KpnI and XhoI (or XhoI /HindIII for CXCR7; all restriction enzymes were acquired from MBI Fermentas), and the amplified CXCR4 and CXCR7 promoter fragments were inserted through ligation. The cloned pGL3-CXCR4 and pGL3-CXCR7 constructs were confirmed by sequencing. The sequentially shorter CXCR4 and CXCR7 promoter fragments were amplified by standard PCR methods, sequenced, and cloned to the pGL3 vector.
Pgl3 basic vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PGL3-basic vector is a plasmid commonly used for gene expression studies. It contains a multiple cloning site for inserting DNA sequences of interest, a bacterial origin of replication, and a selectable marker for antibiotic resistance. The core function of this vector is to provide a platform for cloning and expressing genes in various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Pgl3 basic vector, by Promega (7 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (7 mentions)
Dual luciferase reporter assay system, by Promega (6 mentions)
Dual luciferase reporter system, by Promega (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mutanbest kit, by Takara Bio (1 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (1 mentions)
Dual glo assay kit, by Promega (1 mentions)
Luciferase assay kit, by Promega (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
Dual specific luciferase assay kit, by Promega (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter analysis system, by Promega (1 mentions)
Pgl3 control vector, by Thermo Fisher Scientific (1 mentions)
24 protocols using pgl3 basic vector
1
Cloning and Characterizing CXCR4 and CXCR7 Promoters
2
GPR120 Promoter Regulation Assay
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Seven GPR120 promoter deletion fragments were amplified from pig genomic DNA using primer pairs (Supplement 1, see section on supplementary data given at the end of this article) and inserted into the Kpn I/Xho I sites of pGL3-Basic vector (Fermentas, Promega). The mutants of binding sites were generated using a MutanBEST Kit (Takara, Otsu, Japan) and mutagenic primers (Supplement 1).
All cell lines used were purchased from the China Center for Type Culture Collection (CTCC). 3T3-L1 and pig kidney (PK) cells were fed again with DMEM (Gibco) supplied with 10% fetal bovine serum (FBS, Gibco) at 37 8C with 5% CO 2 . The cells were seeded at a density of 1.5-2.0!10 5 /ml by DMEM supplemented with 10% FBS. After 24 h, they were transfected with plasmid DNA or small interfering RNAs (siRNA) utilizing Lipofectamine 2000 Reagent (Invitrogen).
All cell lines used were purchased from the China Center for Type Culture Collection (CTCC). 3T3-L1 and pig kidney (PK) cells were fed again with DMEM (Gibco) supplied with 10% fetal bovine serum (FBS, Gibco) at 37 8C with 5% CO 2 . The cells were seeded at a density of 1.5-2.0!10 5 /ml by DMEM supplemented with 10% FBS. After 24 h, they were transfected with plasmid DNA or small interfering RNAs (siRNA) utilizing Lipofectamine 2000 Reagent (Invitrogen).
3
Investigating GPX4 3'UTR Regulation by SND1
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The wild-type and truncated mutant 3′UTR of GPX4 were inserted into the pGL3-basic vector (Life Technologies; Thermo Fisher Scientific, Inc.). The empty vector (EV) and SND1 expressing vector (SND1 OV) and pGL3 vector were co-transfected into the cells using Lipofectamine 2000 reagent (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The cells were collected 48 h after transfection and then assayed by the Dual-luciferase reporter assay system (Promega Corporation). The relative firefly activity was normalized to Renilla luciferase activity according to the manufacturer's instructions.
4
Trem1 Promoter Regulation by C/EBPε
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The 253 bp upstream fragment of Trem1 TSS containing the C/EBPε binding site was amplified by PCR using mouse bone marrow mononuclear cells as a template and subcloned into a pGL3-Basic vector (Promega, Madison, WI). The sequence was verified using an ABI PRISM 3100/3130 Genetic Analyzer (Life Technologies). NIH3T3 cells (6 × 105 cells/well in 2 ml DMEM media) were transfected with pcDNA-C/EBPε and the pGL3 basic vector containing wild-type Trem1 promoter sites (TTGTGAAA) or mutated C/EBP binding sites (CCAAGCCG) using Lipofectamine Plus (Life Technologies). Twenty-four hours after transfection, luciferase activities were measured using Promega Dual-Glo assay kit. The Renilla basic vector was cotransfected as a control for normalization.
5
Promoter Activation Assay for Doxorubicin
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Using human genomic DNA (Roche), we amplified 1500 bp upstream of exon 1 using the following primers 5′-CGGCTCGAGGGAGGTGTATGTGAATGAGGTTCC-3′ and 5′-CCCAAGCTTGGGTCCGGAGCCTCCCTCAGACTC-3′. The insert was purified and cloned into a pGL3 basic vector (Life Technologies). Cells were transfected with equal plasmid amount of pGL3 containing insert and a control plasmid containing β-galactosidase. After 24 h, cells were treated with doxorubicin and then assayed for promoter activation using a Luciferase Assay Kit (Promega, Madison, WI, USA). Results were normalized to β-galactosidase activity as measured by a β-galactosidase assay (Agilent).
6
Luciferase Assay for SOX4 and CUL4B
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Cells transfected with indicated plasmid and miRNA mimics/inhibitor were harvested and subjected to luciferase reporter assay using the dual luciferase assay reporter system (Promega). The wild-type and mutant SOX4 3′-UTR vector were constructed from GenePharma (Shanghai). The CUL4B promoter reporter construct was generated by cloning of the promoter region of the gene upstream from the luciferase reporter in the pGL3-basic vector (Life Technologies). Primers for PCR amplification and point mutations were introduced in Supplementary Table 2 . VCaP and HEK293T cells were transfected with the reporter and indicated plasmids or microRNA mimics/inhibitors, and siRNAs. Cell lysates were harvested 48 h after transfection and subjected to luciferase reporter assay using dual-luciferase reporter assay system (Promega).
7
SIRT1 Promoter Regulation by ATF6
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Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) 48 h after RSV treatment according to the manufacturer's protocol. ATF6 in the human SIRT1 promoter region was predicted using three online algorithms, NUBLScan, TRANSFAC, and Gene-Regulation. Based on the over-lapping findings from the predictions, a 1.2-kb human SIRT1 promoter sequence (-1100 to +100 bp) was synthesized and cloned into the XhoI and HindIII sites of the pGL3-Basic vector (Fig. S2C) by Life Technology and then transfected into HepG2 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The resulting construct was confirmed by DNA sequencing. HepG2 cells were transfected with the pGL3-Basic-SIRT1 plasmid and co-transfected with the siRNA for ATF6 or a p3×FLAG-ATF6 plasmid and a pRL-TK plasmid (Promega). Firefly luciferase activity was measured and normalized for transfection efficiency via Renilla luciferase activity. All measurements were performed in triplicate, and the assays were repeated three times in HepG2 cells.
8
Flt3 Regulatory Regions in Luciferase Assay
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The reporter constructs HS-A/HS-B/Luc and HS-A/HS-B/luc/HS-C were generated by cloning the Flt3 regulatory regions at positions -1459bp to +113bp (including HS-A and HS-B) and +7227bp to +8063bp (encompassing HS-C) with respect to the Flt3 ATG, into the pGL3 basic vector (Invitrogen). HPC7 cells were electroporated with 3μg luciferase reporter construct, 2μg expression vector for the MYB or C/EBPα factor or corresponding control vector, and 1μg of β-galactosidase reporter plasmid. Luciferase activities were measured 24h later as previously described [35 (link)]. Luciferase readings were normalised against β-galactosidase activity (Galacton kit, Applera). Identical series of transfection were performed for each separate experiment and results were normalised to 100 to allow comparability of the resulting patterns and minimise artificial variability introduced by the use of different batches of reagents.
9
PLA2G16 Promoter Activity Assay
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The integrated or truncated PLA2G16 promoter segments were cloned in pGL3 basic vector (Promega, Madison, WI, USA). MIA‐PaCa‐2 and PANC‐1 cells were seeded in 24‐well plates at a density of 2 × 105 cells per well. 24 hours later, cells were transfected with either 1 μg of empty pGL3 basic vector or the recombinant vectors with different length of PLA2G16 promoter fragments, using Lipofectamine 3000 (Invitrogen). 0.05 μg of pRL‐CMV vector was co‐transfected to normalize the transfection efficiency. For lentivirus‐infected groups, cells were subjected to lentivirus infection 24 hours after plating and were used for dual‐luciferase assay 24 hours later. After transfection, cells were further cultured for 24 hours. Then, cells were lysed, and the activities of firefly luciferase and renilla luciferase were quantified using a dual‐specific luciferase assay kit according to the manufacturer's instruction (#E1910, Promega), with a luminometer (Promega).
10
Validating miR-506-3p Binding to HOXA11-AS and Slug
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StarBase (http://starbase.sysu.edu.cn/starbase2/ ) and TargetScan (http://www.targetscan.org/vert_71/ ) were used to screen the putative target. Wild-type and mutant type sequences (HOXA11-AS-Wt, HOXA11-AS-Mut, Slug-Wt, Slug-Mut) were constructed and cloned into the pGL3 basic vector (Invitrogen; Thermo Fisher Scientific, Inc.). Huh7 and Hep3B cells were co-transfected with the luciferase vectors and miR-506-3p mimics or miR-NC for 24 h using Lipofectamine 2000 transfection reagent. Luciferase activities were measured by a dual-luciferase assay system (Promega). Firefly luciferase activities were normalized to Renilla luciferase activities.
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