Building on the last experiment, we repeated the Light Cycler test using the Promega S30 kit. We followed the same protocol and the difference now is that we incubated the reaction using a 96-well plate. We also added an additional DNA control of BBa_J364001, otherwise known as Device 2 in the Interlab Study. We used this plasmid because we know it produces visible GFP, but it was not as intense as the Positive control DNA. Having a positive control that is way beyond our signal would make it harder to interpret our results. This data was acquired every 15 minutes instead of every 30 minutes.
The results show a clear difference between the water blank, the master mix only/no DNA control, and the plasmids. The previous results were likely affected by the crushing of the tubes, which is why those water blanks had such high signal. In this experiment, we see that PArsRGFP1.0 duplicates performed the best, PArsRGFP2.0 followed after, and BBa_J364001 was split where one replicate was as bright as PArsRGFP1.0 and the other tracked with PArsRGFP2.0. The No DNA control was lower than all these plasmids, but it was still higher than the water blank. This demonstrates that the master mix does have a lot of autofluorescence.
