On to the Next Phase-- Weeks 4-6 at the Murphy Lab

Hi everyone, I'm Richard and I'll be talking about my past 3 weeks spent at the Murphy Lab, which examines various aspects of the aging process in C. elegans.

Week 3 was a definite struggle.  I ran 3 STAMs with my egl-4 mutants and wild type worms, all of which produced results opposite from what I expected, and since the entire lab was in California at the International C. elegans meeting, I was pretty much on my own when trying to figure out what exactly went wrong.  Geneva hypothesized that the worms were too young, so I made sure to bleach the egl-4 mutants a few hours earlier than I had previously done, and luckily, I was able to confirm my expected result in another STAM.  The rest of the week was spent primarily on preparation for future experiments and analyzing my data.

Week 5 was, for the most part, uneventful.  Originally I had planned to do PCR and run a gel to confirm that my egl-4 crh-1 mating worked to produce double mutants, but since a previous PCR and gel showed that I had no double mutants, I decided it would be best to re-do the original cross. So, once again, the week was spent mostly preparing for the next phase of my project, examining EGL-4::GFP nuclear localization.

For the nuclear localization assay, I first starved my egl-4::GFP worms (these are not the same as egl-4 mutants; rather, they are wild type worms with the EGL-4 protein tagged with GFP so that fluorescence can be observed).  I waited an hour, as I usually do for my chemotaxis assays, and then put the worms on conditioning plates spotted with butanone.  After another hour, I transfered these worms onto a hold plate and prepared microscope slides as quickly as possible to ensure the worms didn't lose their food-odorant association, and then examined fluorescence under a microscope.  To do this, I first locate the head of the worm, at about 60x magnification, and then switch to the option that allows me to view the RFP tagged (red color) AWC neuron.  At this point, the entire neuron, except for the nucleus in the center, flashes a bright red.  I then switch to the option that allows me to view GFP (green color), and depending on whether the nucleus is a bright green or still dark, as observed under RFP, I can tell whether or not the EGL-4 protein has entered the nucleus. I repeated this procedure with naive (untrained) worms, and also with adapted worms (adaptation is when a long, repeated exposure to a certain odor actually diminishes the worms' response to that same odor).  Today I will be analyzing the images I took during these nuclear localization assays.

Overall, I've enjoyed my experience in lab.  However, my days are not always as busy as one would expect, and I've had a couple of setbacks with various aspects of my project, which have consequently resulted in less eventful days.  I guess failure is something every scientist has to deal with sooner or later.