Protocol Online logo
Top : New Forum Archives (2009-): : Cell Biology

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!

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.