A Day in the Yan Lab

After an engaging guest speaker talk in French Science, I begin my (relatively long) journey to the Yan Lab, which is in the Clinical and Research Laboratory building on Research Drive. I arrive at around 10:30 AM (or 9:30 AM on Mondays and Fridays), at which point I begin my day. I start by writing my to-do list for the day, which typically involves some sort of lab maintenance task (such as pouring plates for the worms to live in or washing dishes), a genetic cross for mutant worms, and my two assays for looking at gap junction function in deletion mutant worms. After making my list, I put on my black nitrile gloves and begin my work.

Pouring plates is a relatively long process, so I start with that. It involves making nematode growth medium (NGM), which contains 20 grams of agar mix, 2.5 grams of peptone, and 3 grams of sodium chloride. I add these ingredients, 1 liter of water, and a stir bar to my glass bottles. Then I put the bottles (I make 6 liters) into the autoclave. After an hour in the autoclave, I take the bottles out and put them into a 50°C water bath. I set out 540 petri dishes for pouring, and then I take out the bottles and “dose” them with a few more reagents. I finish the pouring process by pouring about 9.8 mL into each petri dish using a machine.

While I am waiting for the autoclave to finish or the bottles to cool down, however, I am working on my touch assays or crosses. I do two assays to look at gap junction localization phenotypes in worms: imaging with a fluorescent compound microscope and the light touch assay. Imaging involves anesthetizing the worms with 1X M9, putting worms onto a slide, and viewing fluorescent “puncta” (or dots) on the microscope, which signify gap junctions. I record whether each worm’s puncta matches that of the wildtype or of a mutant, and I do this for 50 worms for each strain. The light touch assay requires an unexpected tool: a stick with an eyelash (my eyelash of course) taped to the end of it. Using this tool, I gently tap the worm in the head and the tail 10 times, recording whether or not the worms reacted to the tail touch (because the neurons I am investigating are located in the tail region). I also do this for 50 worms for each strain.

In addition to assaying worms that already carry deletion mutations, I have been working on making new mutant strains through crossing. This involves crossing a mutant worm with a wild-type worm. I will take male wild-type worms and put 10-12 of them on a new plate. Then, I take 2 hermaphrodite worms from a mutant strain and place them on the same plate. I allow these worms to propagate in an incubator, and then I begin my second cross. This cross involves selecting for a specific marker, which is done by selecting worms that fluoresce on the dissecting microscope. After completing this second cross, I allow the worms to propagate, and then I pick 1 worm onto 1 plate for 10 plates in total. These plates will then be genotyped using PCR or Sanger Sequencing to confirm whether they are homozygous or heterozygous.