Firefly Luciferase Assay - variation in FL reading (Apr/24/2009 )

Hello together,

I do in vitro translation with a Firefly Luciferase reporter. Translation efficiency is checked with Firefly Luciferase assay. Unfortunately the variation within 5 readings of FL without any extra treatment gives me quite huge, inacceptable (50%) variation between samples. My values are quite high (10.000-20.000 units). For the test I had one sample and aliquoted in different wells bebfore the reading, so the variation is not due to translation differences.
I guess more variation than 10% is not acceptable. does anyone have ideas or similar experience with FL assay?
As I do not have a Renilla reporter for in vitro translation I cannot normalize, what makes this assay kind of unuseful in this setup. But I guess this variation is not normal, so what could I do wrong?

Thanks a lot for your help.

O yes, I tried homemade buffer as well as commercially available buffer, gives the same pattern/variability, just outputvalues differ in intensity.

Regards
Heiki

I have never actually used this system, but lab colleagues have and they also complain about a large variance within samples. Their proceedure was to take several replicates from the one sample and average the readings.

Alright thanks. Also I think the variation is quite high for an average, it looks more random than anything.

I keep working on it!
Heiki