May 30th, 2007

Lil' Sharkey

::faceplant::

I FINALLY, after a couple months of wanting to cry every time I stepped into lab, figured out why my luciferase assay won't work.

I knew before that it wasn't working because my control Renilla luciferase vector, which is supposed to be what I normalize the firefly luciferase luminescence to, kept giving me lower readings for my experimental condition. Like orders of magnitude lower. It made me cry a little in my heart, and scream a lot out of my mouth.

The reason it doesn't work is stunning. It truly shows the depths of my idiocy.

Firefly luciferase is yellow-green. </i>Renilla</i> luciferase is blue. A different control vector, which I use in place of my experimental vector, has an IRES-EGFP element. The IRES-EGFP element was chopped out of my experimental vector in order to make the vector in the first place.

EGFP is excited by blue light. In fact, in Renilla reniformis itself, EGFP is excited by the light made by Renilla luciferase. It would have been nice if I had looked that up BEFORE, rather than AFTER, my experiments didn't work.

The only good thing about this is that I figured it out before my advisor did.