Transfection - Luciferase

[Arastho]

Hello everyone,

I'm doing some luciferase assays in 293T cells.
In this procedure we had Luciferase plasmid and renilla plasmid as a control.

My question is simple: for reallistic results, the two plasmids should enter on the same cell. In fact, if you are studing a transcription factor (p.e.) that acts on a promotor that regulates the luciferase transcription you should have all the plasmids in the same cell.

Using transfection, how can you ensure that?

Thanks

[kraefzke]

We use EcoTransfect by OZ Biosciences for allmost all transfections in 293 cells. It is cheap, easy to use and very efficient in these cells.
You might have to optimize the ratio of your Plasmids.
Did I understand you correctly, that you want to transfect your cells with a Plasmid encoding a transcription factor and two different Luciferase vectors (Firefly and Renilla, I assume)?

[Arastho]

Originally Posted by kraefzke

We use EcoTransfect by OZ Biosciences for allmost all transfections in 293 cells. It is cheap, easy to use and very efficient in these cells.
You might have to optimize the ratio of your Plasmids.
Did I understand you correctly, that you want to transfect your cells with a Plasmid encoding a transcription factor and two different Luciferase vectors (Firefly and Renilla, I assume)?

Yes, exactly.
Thanks

[kraefzke]

To get the highest number of triple-transfected cells, you should start with the same MOLARITY of all three plasmids. If you encounter difficulties, scale down the concentration of your controll Luciferase (in your case the Renilla plasmid). Try with 10:10:1 for a start and gradually scale down to 50:50:1.
Try not to exceed 5 µg of total DNA per T25 flask or 60mm dish (scale up or down proportionally). You might also have to optimize the ratio of the other two plasmids.
Luciferase assays always worked great for me, with never a problem of getting all the plasmids into the cells (I remember a friend telling me, he once transfected 6 plasmids at once with EcoTransfect into 293 AD cells), so don't worry too much about that part.

[kabdelaz]

Hi, i also have a problem with luciferase transfection, i have tried to transfect it many times to primary cells but i found a difficulty to do that, now iam trying to transfect it to BEAS cells using luciferase plasmid and renilla with ratio 10:1 and lipofectamine as transfecting agent, but i found that all cells are died after 4 hours of transfection, so i will be happy if any one can help me.
Thaqnks
Khaled