LAMP2 3′UTR fragments and mutants containing the miR221 binding site were synthesized and inserted downstream of the luciferase gene in the vector. Mutant (mut) LAMP2 3′UTR was constructed using a site-directed mutagenesis kit (Promega Corporation). The luciferase reporter vector was pGL3 (Shaanxi Youbio Technology Co., Ltd.). 1×105 LX2 cells were transfected with wild-type (wt) LAMP2 3′UTR or mut LAMP2 3′UTR together with miR221 mimics or miR-NC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After transfection for 48 h, a dual-luciferase reporter assay was performed to measure the luciferase activity (Promega Corporation). Luciferase activity was normalized against Renilla luciferase activity (Promega Corporation).
Site directed mutagenesis kit
Manufactured by Promega
The Site-directed mutagenesis kit is a laboratory tool used to introduce specific modifications or mutations into DNA sequences. It provides the necessary components and protocols to efficiently generate desired genetic changes in a targeted manner.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Dual luciferase reporter assay system, by Promega (7 mentions)
Pgl3 control, by Promega (3 mentions)
Pmirglo, by Promega (2 mentions)
Renilla luciferase activity, by Promega (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Dual luciferase reporter kit, by Promega (1 mentions)
Plvx puro vector, by Takara Bio (1 mentions)
Psicheck 2, by Promega (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Pgl3 luciferase reporter vector, by Promega (1 mentions)
Pgl3 control vector, by Promega (1 mentions)
Pmirglo reporter vector, by Promega (1 mentions)
15 protocols using site directed mutagenesis kit
1
Luciferase Assay for miR-221 Targeting LAMP2 3'UTR
2
Investigating miR-149-5p Binding to CDK6
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CDK6 3′-untranslated region (3′UTR) containing the putative binding site of miR-149-5p (CDK6 WT 3′UTR) and the CDK6 3′UTR with the mutation binding site (CDK6 MT 3′UTR) were synthesized by Shanghai GenePharma Co., Ltd. The mutation was generated using a site directed mutagenesis kit (Promega Corporation). The partial sequences of DLEU1 were synthesized by Shanghai GenePharma Co., Ltd. These were then cloned into the vectors (pmirGLO; Promega Corporation). Subsequently, DLEU1 (WT/MT) and CDK6 (WT/MT) were transfected into OSCC cells using Lipofectamine 2000. After 48 h of transfection, the relative luciferase activity was detected using the Dual-Luciferase Reporter kit (Promega Corporation). The data were normalized to Renilla luciferase activity.
3
Generating Constitutive HIF1α Lentivirus
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The human SOD2 expression lentivirus was generated as described previously in our lab [15 (link)]. In order to generate human constitutive HIF1α (HIF1C) lentivirus, the human wild type HIF1α cDNA was obtained from Open Biosystems, and the HIF1α double mutants at P402(A) and P564(A) named as constitutive HIF1α (HIF1C) [25 (link)] were generated using the Site-directed Mutagenesis Kit from Promega. It was then amplified by PCR and subcloned into the pLVX-Puro vector (from Clontech) using restriction sites of BamH I and Xba1 with the following primers: Forward: 5’- tcga-ggatcc – atg gag ggc gcc ggc ggc gcg -3’ (BamH I) and Reverse: 5’- gcgc-tctaga- tca gtt aac ttg atc caa agc -3’ (Xba I), and the HIF1C or empty (CTL) lentivirus was expressed through Lenti-X™ Lentiviral Expression Systems (from Clontech) per manufacturers’ instructions. The virus for HIF1C and SOD2 expression, or related empty (EMP) virus was used to infect THP1 cells, the positive clones were selected by 10μg/ml puromycin, the single colony was picked up, and the expression efficiency for both SOD2 and HIF1C was confirmed by real time PCR and western blotting. The stable SOD2/HIF1C expression THP1 cells were used for in vivo mice xenograft tumor study.
4
AKT3 3'UTR Luciferase Assay
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SKOV3 cells were seeded at 1×105 per well and were serum-starved for 6 h prior to transfection. The RAC-γ serine/threonine-protein kinase (AKT3) 3′-UTR reporter plasmid (500 ng) and the pGL3-control (100 ng) (Promega Corporation, Madison, WI, USA) were co-transfected into cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were harvested and luciferase activities were analyzed after 24 h using the Dual-Luciferase Reporter Assay system (Promega Corporation). Mutants of AKT3 3′-UTR were generated using a Site-Directed Mutagenesis kit (Promega Corporation). The data was normalized by Renilla luciferase activity. The value in miR-NC group was arbitrarily defined as 100%.
5
Regulation of IGF1R by miR-194-5p
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Cells were seeded at 1×105 cells/well and were serum starved for 6 h prior to transfection. The wild-type (WT) 3′UTR of IGF1R and mutated controls were cloned and inserted into the dual-luciferase-reporter plasmid (500 ng; Shengong Biotechnology Company) and the pGL3-control (100 ng; Promega Corporation, Madison, WI, USA). miR-194-5p mimics and controls were subsequently transfected into U373 cells containing wild-type or mutant 3′UTR plasmids with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were harvested and luciferase activities were analyzed after 24 h using the Dual-Luciferase Reporter assay system (Promega Corporation). The mutant IGF1R 3′UTRs were generated using a Site-Directed Mutagenesis kit (Promega Corporation). The primers used to perform mutagenesis were as follows: IGF1R forward, 5′-ctgtccctgatgctgaaggcaggcagaatgacttc-3′, and reverse, 5′-gaagtcattctgctctgccttcagctcagggacag-3′. The kit was used according to the manufacturer's protocol.
6
Regulation of Akt3 by miR-338-3p
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Cells were seeded at 1×105 per well and were serum-starved for 6 h pre-transfection. The 3′untranslated region (3′UTR) of Akt3 and mutated controls were cloned and inserted into the reporter plasmid (500 ng) and the pGL3-control (100 ng; Promega, Madison, WI, USA). MiR-338-3p p mimics were then transfected into the Caki-1 cells containing the wild-type or mutant 3′UTR plasmids with Lipofectamine 2000 (Invitrogen). Cells were harvested and luciferase activitity was measured after 24 h using the Dual-Luciferase Reporter Assay System (Huijun Company, Guangzhou, China). Mutant of Akt3 3′UTR were generated using the Site-Directed Mutagenesis kit (Promega).
7
Regulation of MSI2 Promoter Activity
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A549 and H520 cells were transfected with MSI2 promoter reporters, si-KLF4-2, or pKLF4. The mutation was generated using a site directed mutagenesis kit (Promega Corporation). The partial sequences of KLF4 and MSI2 were synthesized by Shanghai GenePharma Co., Ltd. These were then cloned into the vectors (pmirGLO; Promega Corporation). Subsequently, pKLF4/si-KLF-4-2 and pMSI2 were co-transfected into NSCLC cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The activity of the MSI2 promoter was normalised by co-transfection with a β-actin/Renilla luciferase reporter containing the full-length Renilla luciferase gene. The intensity of luciferase activity was quantified after 24 h of transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation).
8
Regulatory Interaction of miR-1225-5p and Sox9 in NSCLC
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The target genes of miR-1225-5p were predicted using miRanda (http://www.microrna.org ) (19 (link)). To confirm the interaction between miR-1225-5p and Sox9 in NSCLC cells, 4x104 A549 and H1299 cells/well were plated into six-well plates and cultured at 37˚C for 24 h. Subsequently, the wild-type (WT) or mutant (MUT) 3'-UTR of Sox9 was cloned into the luciferase reporter vector PsiCheck2 (Promega Corporation). The MUT sequence was generated using a site-directed mutagenesis kit (Promega Corporation). The WT or MUT Sox9 luciferase plasmid (100 ng) was co-transfected into A549 and H1299 cells with the miR-1225-5p mimic (50 nM), miR-1225-5p inhibitor (100 nM) or the respective NCs (50 nM mimic NC; 100 nM inhibitor NC) using Lipofectamine 2000, according to the manufacturer's protocol. Following incubation for 48 h at 37˚C, the relative luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
9
Plasmid-based Manipulation of SUN1 and Nesprin-2 in HeLa Cells
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Plasmids encoding human full length SUN1 (GFP-SUN1) and the siRNA resistant (R) GFP-SUN1R as well as GST-SUN1-NT (residues 1-239) have been described previously (SUN1 Accession number O94901)8 (link). Mutations to generate GFP-SUN1 or GFP-SUN1R S110A, S113A, S110A/113A and S113D and GST-SUN1-NT S110D, S113D, S110D/S113D were introduced using a site-directed mutagenesis kit (Promega). GFP-SUN1R-ΔC contains residues 1 to 412 encompassing the N-terminus and the transmembrane region but lacking the coiled coil region and the SUN domain. Transfections of HeLa cells were carried out using Lipofectamine 2000 (Invitrogen) or o-fekt (Bio-Budget Technologies, Krefeld, Germany). Analyses were done 24 to 48 hours after transfection. For siRNA-mediated depletion, HeLa cells were transfected with SUN1 targeted siRNA or control siRNA described previously8 (link) and SUN2 targeted siRNA (GE Healthcare Dharmacon). For RNAi transfection, the reagent INTERFERin (Polyplus) was used. Cells were analyzed 72 to 96 hours after transfection. For rescue experiments, cells were transfected with GFP-SUN1R or GFP-SUN1R-ΔC plasmids using Lipofectamine 24 h after siRNA treatment. Analyses were done 72 to 96 hours after siRNA treatment8 (link).
sh RNA knockdown of Nesprin-2 was carried out using oligonucleotides targeting N- and C-terminal sequences of Nesprin-2 Giant as described19 (link), 20 (link).
sh RNA knockdown of Nesprin-2 was carried out using oligonucleotides targeting N- and C-terminal sequences of Nesprin-2 Giant as described19 (link), 20 (link).
10
Validating miR-15a-5p Binding Sites
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miR-15a-5p binding sites were identified by StarBase database (http://starbase.sysu.edu.cn ). The dual luciferase reporter assay was performed according to a previous study (36 (link)). ANRIL and JAK2 3′-UTRs containing the putative binding sites (GCUGCU) of miR-15a-5p were synthetized and obtained from Sangon Biotech Co., Ltd. The aforementioned sequences were cloned into the pmirGLO vector (Promega Corporation) to construct wild-type (WT) or mutant (MUT) ANRIL (10 nM) and JAK2 reporter vectors (10 nM). Point mutations of the miR-15a-5p binding sites were generated using a Site-Directed Mutagenesis kit (Promega Corporation). The WT or MUT ANRIL/JAK2 vectors (10 nM) were co-transfected into HNECs (5×106 per well) with mimics NC or miR-15a-5p mimics (10 nM) using Lipofectamine 2000 reagent for 24 h at 37°C, according to the manufacturer's instructions. After 24 h, relative luciferase activities were detected using a Dual-GLO Luciferase assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
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