The nanoluciferase-based MMEJ assay was carried out following a similar protocol to what has been previously described (22 (link)). Briefly, the Nano-luciferase MMEJ reporter construct (a linearized plasmid with protruding microhomology ssDNA ends engineered such to recombine into a functional luciferase construct only after correctly performed MMEJ) (22 (link)) was transfected into cells together with a control firefly luciferase plasmid. Transfected cells were plated into 384 or 96-well plates already containing diluted compound and luminescence was read 24 h after transfection. Firefly and Nano-luciferase levels were detected using the Nano-Glo Dual-Luciferase Reporter Assay kit (Promega) and luminescence was measured with a Clariostar plate reader (BMG Labtech). The nanoluciferase signal was normalized to the firefly luciferase signal to correct for cell density and transfection efficiency, and finally normalized to vehicle treated controls.
Nano glo dual luciferase reporter assay kit
Manufactured by Promega
The Nano-Glo Dual-Luciferase Reporter Assay kit is a luminescent reporter assay system that measures the activities of two different luciferase enzymes simultaneously within a single sample. The kit provides the necessary reagents to perform dual-reporter gene analyses.
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Lab products found in correlation
3 protocols using nano glo dual luciferase reporter assay kit
1
High-Throughput MMEJ Repair Assay
2
Notch Signaling Activation Assay
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HS5 cells were transiently transfected with a Notch reporter plasmid pNL2.1 carrying a 6xCSL Notch responsive element26 (link) and with the vector constitutively expressing the firefly luciferase upon the thymidine kinase promoter (pGL4.54[luc2/TK]). After 24 hours (h), HS5 cells were cultured alone or placed in co-culture with scrambled (Scr) or Jagged1 and Jagged2 knockdown (J1/2KD) HMCL and incubated for 24 h. Luciferase activity was measured using Nano-Glo® Dual-Luciferase® Reporter assay kit (Promega) on the Glowmax instrument (Promega).
3
Microhomology-Mediated End Joining Assay
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The nanoluciferase-based MMEJ assay was carried out following a similar protocol to what has been described previously (22 (link)). Briefly, the Nano-luciferase MMEJ reporter construct (a linearized plasmid with protruding microhomology ssDNA ends engineered such to recombine into a functional luciferase construct only after correctly performed MMEJ; ref. 22 (link)) was transfected into cells together with a control firefly luciferase plasmid. Transfected cells were plated into 384- or 96-well plates already containing diluted compound and luminescence was read 24 hours after transfection. Firefly and nano-luciferase levels were detected using the Nano-Glo Dual-Luciferase Reporter Assay Kit (Promega) and luminescence was measured with a Clariostar plate reader (BMG Labtech). The nanoluciferase signal was normalized to the firefly luciferase signal to correct for cell density and transfection efficiency, and finally normalized to vehicle-treated controls.
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