Luciferase assay - (May/12/2011 )

Hi, I have been doing luciferase assays with the dual-Glo luciferase assay system from Promega for quite some time using the Perkin Elmer wallac 1420 multilabel counter, and my mock transfected cells always have a very low raw reading ~300, however recently, I noticed my protocol has been accidentally changed by someone in the lab from a reading of 5 seconds to 1 second, I have changed it back and checked the protocol against one that was done a long time ago and there doesn't seem to be any differences. However, ever since that day (including the reading that was done with the 1 second), my background raw reading is very high(~3800), and my transfected well reading has not gone up proportionally. I have tested wells with no cells and also using another batch of reagents from another lab, but the blank reading is still very high. I have also checked the counter itself to see if there are any physical differences, and I have asked others if they noticed anything different with their recent results, but there doesn't seem to be any.....I am trying to finish my PhD and I am getting a little desperate...

Does anyone know or have experienced anything that might explain what is wrong?

Many thanks!

If you read a completely empty plate, does the background change from the first to the last well?

I just tried reading an empty plate yesterday and the readings are nearly identical to my previous wells with reagents only and mock transfected wells, and no, the values do not change from the first to last....
I have no idea where the reading is coming from....I asked other lab members again if they notice something with their readings (although they are not doing luciferase) as I thought if there is something funny with the machine then it will apply for everyone, but others seem to be fine with their numbers

96well on Mon May 16 11:52:46 2011 said: