Because we wanted to increase the sensitivity of GFP detection, we used a Roche Light Cycler 480 to monitor the formation of GFP over time. We needed to use the Light Cycler because we can control the temperature and monitor the reaction in real-time, something that our plate reader is not able to do. The manufacturer’s protocol was followed as before, but this time we increased the DNA concentration to 30ng/uL and the reaction volume was 25uL in individual PCR tubes. We only tested the GFP producing plasmid. The reaction was incubated at a constant 37C and we took an acquisition at the start of the cycle, and then once every 30mins.
This data looks more encouraging. It appears that PArsRGFP2.0 performed better than PArsRGFP1.0, and these were better than the no DNA controls. It is unusual that the water blanks had signal. Overall, the signal increase is low, but it is a start.
