Help with Dual luciferase assay - (Mar/07/2012 )

I am trying to do a dual luciferase assay using Promega Dual luciferase kit. I have plated 50K and 100K cells in 6 well plates. I am transfecting in a total of 1000 ng of DNA. I have done testing to determine the amounts of each plasmid and when I am putting all of the them together I get random crazy readings.

I am transfecting in 100 ng of reporter which is HLA-DR (MHC II) luc, 3.3 ng of renilla, varying concentrations of my proteins of interest.

The values that I get over the (now 8 times) I have done this stupid assay are different every time. For a long time I was getting VERY large renilla values...and low luciferase numbers. I have made all new stock of my plasmids and grown new cells. Today when I repeat this assay, my number for renilla are lower(from the hundreds to 1.5....) but my Luciferase readings are also very low. When I try to graph the data I am dividing the luciferase/renilla values. Basically it showed all the same trend no matter what I put in...and I know that can not be correct.

I don't understand why when I have done dual luciferase assays a million times with more DNA than this, that now it is not working.

I am using the Softmax Pro software and a plate reader. I have it set up to integrate @ 10 sec with each injector set to 100uL with a 2 sec delay.

Any help would be greatly appreciated. Please let me know if more information is needed...I just am not sure what to write to get help!

DESPERATELY WANT TO GRADUATE!!!
Jewels :-)

I had the exact same problem. Done an assay a million times and then one day it just stopped working. I still don't have an explanation to the problem but it helped when I used new cells and changed transfection reagent and this also meant that I had to re-optimize the assay.
Just do a simple experiment only using the renilla construct. Transfection with increasing amounts of renilla and determine the level where it reaches the saturation point (e.g increasing renilla= increasing signal to a point where it only levels off). Do this with all the construct and finally do the experiment with the amount of DNA that does saturate the signal.