The wild-type or mutant 3′-UTR of TLR4 was cloned into the pmiRGLO vector (Promega Corporation). The recombinant plasmids were acquired using the EndoFree Plasmid Maxi Kit (Vazyme). The cells were seeded in 24-well plates and co-transfected with miR-182-5p mimic or mimic control and the MUT or WT 3′-UTR sequences of TLR4 using Fugene transfection reagent (Promega Corporation) for 48 h. The Renilla luciferase pRL-TK vector was used as a control. Following transfection, the cells were incubated for 48 h and Renilla luciferase activity was tested using the dual-luciferase reporter assay (Promega Corporation) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.
Endofree plasmid maxi kit
Manufactured by Vazyme
The EndoFree Plasmid Maxi kit is a laboratory product designed for the purification of plasmid DNA. It utilizes an optimized protocol to efficiently isolate high-quality plasmid DNA from bacterial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Pmirglo vector, by Promega (6 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Dual luciferase reporter assay, by Promega (2 mentions)
Fugene transfection reagent, by Promega (2 mentions)
Prl tk renilla luciferase vector, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
6 protocols using endofree plasmid maxi kit
1
Regulation of TLR4 by miR-182-5p
2
Exploring miR-1297 and FA2H Interaction
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Bioinformatics analysis was performed using TargetScan 7.2 (http://www.targetscan.org/vert_72/ ). And the results showed the potential binding sites between miR-1297 and the 3′-UTR of FA2H. The wild-type (WT) or mutant (MUT) 3′-untranslated regions (3′-UTRs) of FA2H were cloned into the pmiRGLO vector (Promega Corporation). The recombinant plasmids were acquired using an EndoFree Plasmid Maxi kit (Vazyme Biotech Co., Ltd.). Subsequently, 293T cells (5×104 cells/well; American Type Culture Collection) were seeded into 24-well plates and co-transfected with 50 nM miR-1297 mimic (cat. no. MCH01244; Applied Biological Materials, Inc.) or 50 nM mimic control (cat. no. MCH00000; Applied Biological Materials, Inc.) and 1 ng MUT FA2H 3′UTR or 1 ng WT FA2H 3′UTR using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. The pRL-TK plasmid (Promega Corporation) containing the Renilla luciferase gene was u firefly sed as an internal control. At 48 h post-transfection, and Renilla luciferase activities were detected using the Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity. Besides, 293T cells were transfected with miR-1297 mimic or mimic control for 48 h, and the transfection efficiency was confirmed using qRT-PCR.
3
Regulation of FA2H by miR-300
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The wild-type (WT) or mutant (MUT) 3′-untranslated region (3′-UTR) of FA2H was cloned into a pmiRGLO vector (Promega Corporation). Recombinant plasmids were acquired using an EndoFree Plasmid Maxi kit (Vazyme Biotech Co., Ltd). A total of 293 T cells seeded in 24-well plates were cotransfected with miR-300 mimics (sense: 5′-UAUACAAGGGCAGACUCUCUCU-3′, antisense: 5′-AGAGAGUCUGC CCUUGUAUAUU-3′; GenePharma) or negative control (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGG AGAATT-3′; GenePharma) and the MUT or WT 3′-UTR of FA2H for 48 h together with renilla luciferase pRL-TK vector as a control. Following transfection for 48 h, firefly and renilla luciferase activities were tested using a dual-luciferase reporter assay (Promega Corporation). Firefly luciferase activity was normalized to renilla luciferase activity.
4
Characterization of miR-361-5p and MAP3K9
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Bioinformatics software analysis (TargetScan version 7.2; http://www.targetscan.org/vert_72/ ) was used to predict the potential targets of miR-361-5p. The results revealed the potential binding sites between miR-361-5p and MAP3K9. To confirm this, the wild-type (wt) or mutant (mut) 3'untranslated region (UTR) of MAP3K9 was cloned into the pmiRGLO vector (Promega Corporation). The recombinant plasmids were acquired using EndoFree Plasmid Maxi kit (Vazyme Biotech Co., Ltd.). Huh7 cells seeded (5x104 cells per well) in 24-well plates were co-transfected with miR-361-5p mimics or negative control and the mut or wt 3'-UTR of MAP3K9, together with the Renilla luciferase pRL-TK vector as a control. After transfection at 37˚C for 48 h, firefly and Renilla luciferase activity was measured using a dual-luciferase reporter assay kit (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
5
Regulation of HOTAIR 3'UTR by miR-126
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The wild-type (WT) or mutant (MUT) 3' untranslated region (UTR) of HOTAIR was cloned into the pmiRGLO vector (Promega Corporation). Recombinant plasmids were acquired using the EndoFree Plasmid Maxi kit (Vazyme Biotech Co., Ltd.). HaCaT cells (5x104 cells per well) were co-transfected with 100 nM miR-126 mimic or 100 nM mimic control and 1 ng MUT-3'UTR-HOTAIR or 1 ng WT-3'UTR-HOTAIR at 37˚C for 48 h. Renilla luciferase pRL-TK vector (Promega Corporation) was used as the control. At 48 h post-transfection, luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
6
Targeting TLR4 by miR-489-3p
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Bioinformatics analysis using TargetScan 7.2 (http://www.targetscan.org/vert_72/ ) was performed to determine the binding sites between miR-489-3p and TLR4. The wild-type (WT) or mutant (MUT) 3'UTR of TLR4 was cloned into the pmiRGLO vector (Promega Corporation) and the recombinant plasmids were acquired using an EndoFree Plasmid Maxi kit (Vazyme Biotech Co., Ltd.). To point-mutate the miR-489-3p binding domain in the 3'UTR of TLR4, a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.) was used according to the manufacturer's instructions. Cells were seeded into 24-well plates at a density of 5x104 cells/well and co-transfected with a miR-489-3p mimic or mimic control and the MUT or WT 3'UTR of TLR4 using Fugene transfection reagent (Promega Corporation) according to the manufacturer's protocol, together with the Renilla luciferase pRL-TK vector (Promega Corporation) as a control. Following transfection at 37˚C for 48 h, firefly and Renilla luciferase activities were determined using a Dual-Luciferase Reporter assay system (Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.
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