Hello
I'm trying to do the luciferase assay with my manipulated vector but I'm getting very low luciferase signal. The positive controls for luciferase and renilla are OK and i'm getting a high intensity signal. The GFP C+ is OK means that the transfection is OK. I have checked also un-manipulated (Original) vector, but I have got the same results. Is anyone have any suggestion? My lucia vector concentration was 100 ng per well co-transfected with 2 and 10 ng of Renilla.
Best Regards
Hi,
are yo uusing a minimal promoter Luciferase reporter? Might it be that your promoter sequence is not working in these cells? This would explain why the empty vector (I assume this is the non-manipulated vector) is also giving low singals.
Sven
Hi Sven,
Thank you for your response, The promoter is OK. Its an hEG1 Promoter and I have checked it by sequencing.
Majid
Kevin Kopish
Hi, Thanks a lot for your response. Actually, I have found the problem. Based on the vector features the Lucia is releasing to the medium. so I used another kit from Invivogene to measure the Lucia from the medium named QUANTI-Luc™ that worked very well. But to measure the Renilla I need again the Dual-Glo of Promega. It seems you are working in Promega, so if you have any product that can help me, would be nice to let me know.
Best
Kevin Kopish
Hi kevin, I have done another experiment and transfect my cells with and without Renilla. The results shows that there is no interaction between Lucia and Renilla. I found that one of the main issues with your Kits is the lysis power of the buffer in E2920 . After even 30 min of incubation and pipetting there are many cells remained intact. When I have used another lysis buffer prepared by myself, It worked very well in less than 5 min. This problem will lead to variation in the cell lysis and eventually in Renilla measures.
Thanks for your answer.
Best
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