Posted 12 May 2011 - 03:37 AM
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?