Luciferase reporter vector pmirGLO with the full length of the 3′-UTR of E-cadherin or circ-AKT3 were constructed. Then we generated the mutant luciferase reporter vectors with QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, California, USA). OSRC-2 and SW839 cells were seeded into 12-well plates and transfected with luciferase reporter vector and miR-296-3p mimics using the Lipofectamine 3000 reagent and Lipofectamine RNAiMAX reagent respectively. After 48 h incubation, the Firefly and Renilla luciferase activities were quantified with a dual-luciferase reporter assay (Promega, USA) according to the manufacturer’s protocol.
Xl site directed mutagenesis kit
Manufactured by Qiagen
Sourced in United States, Germany
The QIAGEN XL-site directed Mutagenesis Kit is a tool designed for efficient and precise genetic modifications. It enables the introduction of specific mutations, insertions, or deletions into DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Prl tk, by Promega (5 mentions)
Pgl3 control vector, by Promega (5 mentions)
Dual luciferase assay, by Promega (4 mentions)
Dual luciferase reporter assay, by Promega (3 mentions)
Dual luciferase reporter assay kit, by Promega (3 mentions)
Psicheck 2 luciferase vector, by Promega (2 mentions)
Firefly luciferase reporter vector, by Promega (1 mentions)
Amaxa nucleofector, by Lonza (1 mentions)
Dual glow luciferase assay, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pmirglo, by Promega (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pmirglo, by Thermo Fisher Scientific (1 mentions)
Pmirglo vector, by Promega (1 mentions)
13 protocols using xl site directed mutagenesis kit
1
Luciferase Assay for miRNA Targeting
2
LDHA 3'UTR Regulation by miR-34a
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The full-length of the 3′-UTR of LDHA was amplified by PCR from MDA-MB-231 genomic DNA and inserted into pGL3 control vector (Promega, WI). Using the QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, CA), we generated several inserts by deletions of 4 bp from the perfectly complementary site of the LDHA gene. According to the manufacturer’s instructions (solution V, program T-016), MDA-MB-231 cells were cotransfected with 0.5 μg firefly luciferase report vector and 0.5 μg control vector containing Renilla luciferase, pRL-TK (Promega) by nucleoporation (AmaxaBiosystems). Each nucleoporation used 50 nM of the miR-34a mimics or scrambled oligonucleotide. 48 hours after transfection, the relative luciferase activities (RLA) of firefly and Renilla were consecutively measured using the dual luciferase assay (Promega).
3
Functional Validation of miR-124 Regulation of SP1
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The sequence of the 3′ untranslated region (UTRs) of the SP1 gene was obtained from the TargetScan database version 7.1 (http://www.targetscan.org/vert_71/ ). The 3′UTR was amplified by PCR (14 (link)) from the genomic DNA of the SGC-7901 cell line and inserted into a pGL3 control vector (Promega Corporation) using the XBA1 site immediately downstream from the stop codon of luciferase. The SP1 primer sets used for PCR were as follows: Forward 5′-CCTTCAGGGATTTCCAACTG-3′ and reverse, 5′-GTCCAAAAGGCATCAGGGTA-3′. A mutant insert was also generated in which the first four nucleotides of the miR-124 binding site the SP1 gene (AUGT-GCAC; predicted by TargetScan) were mutated using a QIAGEN XL-site directed Mutagenesis kit (Qiagen, Inc. Valencia, CA, USA). SGC-7901 and MKN-28 cells were cotransfected by nucleoporation (Amaxa Nucleofector™; Lonza Group, Ltd., Basel, Switzerland) (16 (link)) with 5 µg firefly luciferase reporter vector (Promega Corporation) and 0.5 μg control vector containing Renilla luciferase (pRL-TK; Promega Corporation). For each nucleoporation, 50 nM of the miR-124-3p mimic, miR-124-LNA (inhibitor), miR-ctr or miR-LNA was used. Firefly and Renilla luciferase activities were measured consecutively using a dual luciferase assay (Promega Corporation) at 48 h after transfection.
4
Investigating KRAS 3'-UTR Regulation by miR-200c
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The full-length of 3′-UTRs of the KRAS gene was amplified by PCR from MDA-MB-231 genomic DNA and inserted into pGL3 control vector (Promega, WI). Using the QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, CA), we generated several inserts by deletions of 4 bp from the perfectly complementarity site of KRAS gene. According to the manufacturer's instructions (solution V, programme T-016), MDA-MB-231 cells were cotransfected with 0.5 ug firefly luciferase report vector and 0.5 ug control vector containing Renilla luciferase, pRL-TK (Promega) by nucleoporation(AmaxaBiosystems). Each nucleoporation used 50 nM of the miR-200c or a scrambled oligonucleotide. 48 hours after transfection, the relative luciferase activities (RLA) of Firefly and Renilla were consecutively measured through the dual luciferase assay (Promega).
5
Regulation of LDHA Expression by miR-383
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The full-length of LDHA 3′-UTR was amplified and inserted into pGL3 control vector (Promega, WI). Then we generated mutations by deletion of 4 bp from the perfectly complementary site of the LDHA 3′-UTR using the QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, CA). Next, we co-transfected HepG2/SMMC-7721 cells with 0.5 µg firefly luciferase report vector and 0.5 µg control vector containing Renilla luciferase, pRL-TK (Promega), followed by 50 nM miR-383 mimics or scrambled oligonucleotide. Finally, the relative luciferase activities (RLA) were measured 48 hr after transfection using the dual luciferase assay (Promega).
6
Regulation of MCL1 by miR-125b
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MGC-803 cells (6 × 104) were seeded in 24-well plates immediately prior to transfection. The pMIR-MCL1 plasmids were transfected into MGC-803 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. We also generated several inserts with deletions of 4 bp from the site of perfect complementarity of the MCL1 gene using the QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, Valencia, CA). The miR-125b mimics and pMIR-MCL1 plasmids were cotransfected where indicated. Forty-eight hours after transfection, cells were assayed for both firefly and Renilla luciferase using the dual luciferase glow assay (Promega). Transfection experiments were performed in duplicate and repeated at least three times in independent experiments.
7
Luciferase Assay for miR-346 Regulation
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Luciferase reporter vector with the full length of the 3′-UTR of FBLIM1 or circFBLIM1 were constructed. Then we generated the mutant luciferase reporter vectors with QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, California, USA). HepG2 cells were seeded into 96-well plates and co-transfected with luciferase reporter vector and miR-346 mimics or miR-346 LNA using the Lipofectamine 2000 transfection reagent. After 48 h of incubation, the firefly and Renilla luciferase activities were quantified with a dual-luciferase reporter assay (Promega, USA).
8
Luciferase Reporter Assay for 3'-UTR Interactions
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Luciferase reporter vector with the full length of the 3'-UTR of SLC7A11 or circ0097009 were constructed. Then we generated the mutant luciferase reporter vectors with QIAGEN XL-site directed Mutagenesis Kit (QIAGEN, California, USA). HepG2 cells were seeded into 96-well plates and co-transfected with a luciferase reporter vector and miR-1261 mimics or miR-1261 LNA. After 48 h of incubation, the luciferase activities were quanti ed with a dual-luciferase reporter assay (Promega, USA). Cells (5×10 3 ) were plated and co-transfected with the constructed vectors and miR-1261 mimics for incubation (48 h). Then, the relative luciferase activity was assessed by a dual-luciferase reporter assay system (Promega).
9
miR-143 Regulation of MAPK1 3'UTR
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MAPK1 3’UTR with miR-143 binding site was amplified to psiCHECK-2 luciferase vector (Promega, Madison, WI, USA) as luc-MAPK1 vector. T Luc-MAPK1-mut vector with MAPK1 3’UTR mutation without miR-143 binding site was constructed by XL Site-directed Mutagenesis Kit (Qiagen, Hilden, Germany). Cells were co-transfected with Luc-MAPK1-wt/Luc-MAPK1-mut and miR-143/negative. After transfected for 48 h, cell gene activity was tested by dual luciferase assay kit (Promega, Wisconsin, USA). Turner Designs Spreadsheet Interface Version 2.0.1 was used for calculation and analysis.
10
Regulation of NRAS 3' UTR by miR-145-5p
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XL Site‐directed Mutagenesis Kit (Qiagen, Germany) was used to construct the mutated 3' UTR of NRAS which did not contain the binding sites of miR‐145‐5p. The normal or mutant 3' UTR sequences of NRAS were inserted into psiCHECK‐2 luciferase vectors (Promega), respectively. After successfully constructed, the Luc‐NRAS and Luc‐NRAS‐mut vectors were transected to the 293T cells together with miR‐145‐5p mimics or mimics control (diagnosed as NC group) using liposome 2000 kit (Invitrogen). The relative luciferase activities were measured using Dual‐Luciferase Reporter Assay Kit (Promega) in strict accordance with the manufacturer's instructions.
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