pSG5-FER (human FER tyrosine kinase) plasmid was purchased from Addgene (Cambridge, MA) and propagated in E. coli. Plasmids were then harvested using QIAGEN Giga-prep kits (Valencia, CA)(54 (link)) per manufacturer’s instructions. Original pSG5-FER plasmid was a gift from Nora Heisterkamp deposited in Addgene DNA repository (Addgene plasmid # 30191)(54 (link)). The plasmid contains an insert that encodes human FER (hFER) which is a non-receptor tyrosine kinase located on human chromosome 5q21 (Gene ID 2241; HPRD 01491; NM_005246). Two separate plasmids containing the α and β subunits of the Na+/K+-ATPase were a gift from David A. Dean (University of Rochester, NY) and were harvested in similar fashion as described above. A luciferase reporter gene plasmid (Promega, Madison, WI) was used as a control for effects of electroporation process over results, containing similar promoter sequence of the FER plasmid (SV40). Similarly an empty plasmid (pcDNA3) was used for same purposes in survival curve experiments. All plasmids of DNA were stored in 10 mM Tris- 1 mM EDTA buffer (pH 8.0) at −20°C until the day of gene delivery. Protocols for the use of recombinant DNA technology required prior approval from the University of Michigan Institutional Biosafety Committee.
Luciferase reporter gene plasmid
Manufactured by Promega
Sourced in United States
The Luciferase reporter gene plasmid is a laboratory tool used to measure gene expression. It contains a luciferase gene, which produces a bioluminescent signal when expressed. This signal can be quantified to infer the activity of a promoter or regulatory sequence linked to the luciferase gene. The plasmid provides a standardized platform for studying gene regulation and monitoring cellular processes.
Automatically generated - may contain errors
4 protocols using luciferase reporter gene plasmid
1
Plasmid Purification and Propagation
2
Plasmid Purification and Propagation
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pSG5-FER (human FER tyrosine kinase) plasmid was purchased from Addgene (Cambridge, MA) and propagated in E. coli. Plasmids were then harvested using QIAGEN Giga-prep kits (Valencia, CA)(54 (link)) per manufacturer’s instructions. Original pSG5-FER plasmid was a gift from Nora Heisterkamp deposited in Addgene DNA repository (Addgene plasmid # 30191)(54 (link)). The plasmid contains an insert that encodes human FER (hFER) which is a non-receptor tyrosine kinase located on human chromosome 5q21 (Gene ID 2241; HPRD 01491; NM_005246). Two separate plasmids containing the α and β subunits of the Na+/K+-ATPase were a gift from David A. Dean (University of Rochester, NY) and were harvested in similar fashion as described above. A luciferase reporter gene plasmid (Promega, Madison, WI) was used as a control for effects of electroporation process over results, containing similar promoter sequence of the FER plasmid (SV40). Similarly an empty plasmid (pcDNA3) was used for same purposes in survival curve experiments. All plasmids of DNA were stored in 10 mM Tris- 1 mM EDTA buffer (pH 8.0) at −20°C until the day of gene delivery. Protocols for the use of recombinant DNA technology required prior approval from the University of Michigan Institutional Biosafety Committee.
3
Validating miR-1839-5p Targets Ier2/TSPO
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Luciferase reporter gene plasmids (Promega Corporation) containing the 3′-UTR of Ier2/TSPO with predicted miR-1839–5p target sites (wild-type) or mutated sites for the 3′UTR of Ier2/TSPO (Mutant) were constructed (Hanbio, Shanghai, China). Lipofectamine 2000 (Invitrogen) was used to co-transform cells with the miR-1839–5p mimic and lncRNA Ier2 WT/Mut or miR-1839–5p mimic and TSPO WT/Mut for 48 h. After co-transformation 48 h, the cells were collected using 1xPassive Lysis Buffer, and centrifuged (12000 rpm) at 4 °C for 10 min. The supernatant was subsequently collected, and the firefly luciferase activity was assayed and normalized to Renilla luciferase activity according to the manufacturer's instructions (Promega).
4
Luciferase Assay for miRNA-Target Interaction
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The luciferase reporter gene plasmids (Promega Corporation, Madison, WI, USA) containing the 3’-UTR of PIM-2 with predicted miR-24-3p target sites (Wild-type) or the mutated sites of 3’UTR of PIM-2 (Mutant) were constructed. HEK 293T cells were then transiently co-transfected with the above luciferase reporter gene plasmids and miR-24-3p mimic (miR-24-3p) or negative control., Firefly and renilla luciferase activities were measured 48 h after co-transfection using a dual-luciferase reporter assay system (Promega Corporation).
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