Hot and cold!
- I ran a restriction to check whether the mutated red luciferase with mutated RBS restriction was present. The last two lanes indicate that the product has the expected size.
Click beetle luciferases assay
- Today, I also used the cultures grown overnight to check whether the luciferases showed any brightness. I followed the assay mentioned by Edinburgh's team for two cultures of green emitting luciferase and two cultures of red. To that, I added 1μL of 100mM luciferin and waited in the dark room for ~30min taking pictures with a SLR camera with high ISO (~400), for about 30secs to 1 min exposure. I observed nothing :'(.
- After the initial disappointment, I talk to Cambridge team and they help us while trying to understand the causes why the assay was a complete disaster. I will continue to check the mutated luciferase from firefly along with the pT7 luciferase.