Transfection - beginner - (Aug/02/2006 )

Hello everyone!
I'm starting with MCF-7 transfection. I just want to compare luciferase (firefly) intensity of my different vectors, i will make a co-transfection with a vector with renilla luciferase and use the dual-luciferase from promega. I have some doubts about transfection efficiency, and I saw I have to use a GFP vector. Is this the part when I have to optimize the amount of DNA to further use with my experimental vectors? In that case I have to choose the same plate/dish... because i just need to measure luciferase intensity i can use a 12 or 24 well plate or it is necessary to have more cells?

Thanks, it is my first time here... I hope you can help me... more questions will come soon...

Virgínia

No you dont have to use EGFP. You will get Firefly luciferase reading as your promoter activities and Renilla luciferase activity to normalize, or correct your transfection of cells in different wells. You can always compare activity differences among cells transfected.

You can optimize transfection conditions with luciferase activity as the end point, using fixed amount of DNA (0.1-0.2ug/well for 96 well plate, and twicw that for 48 well plate), and varied amount of liposome or polymer transfection reagent as recommended by the supplier.

You can use 48, or even 96 well plates as you are using a very sensitive reporter system.

As it is indeed a very sensitive reporter system, make sure you get the right plates (not transparant ones, you will get spill-over readings).
Maybe shouldn't give the hint at all, but an ex-colleague had very positive results once...

Thanks a lot! It was very heplfull

Best

Virgínia