PROTOCOL: -amanitin treatment of starved L1 larvae

5 December 2011 Modified 30.05.2012 Michael Lab

CAUTION: -amanitin comes from the death cap mushroom and is one of the most infamous human poisons of all time. Please handle it according. This protocol is designed such that powdered -amanitin is never released into the atmosphere.

Prepare -amanitin solution: -amanitin (Sigma A2263) is sold in a 1mg unit in a rubber stopper bottle. The final solution should be made to 1mg/mL in water. Carefully measure 1mL of water into a sterile 1.7mL tube. Use a sterile syringe to then inject the water through the rubber stopper directly into the brown bottle. Once the powder is dissolved, a sterile needle and syringe can be used to withdraw an aliquot of the solution into a 1.7mL tube as a working stock (0.1mL or so at a time). Store the -amanitin solutions in the metal can in the -20C freezer. Day 1: Isolate embryos from >5 plates of gravid adults and hatch overnight in 25mL 1X M9 salts. Day 2: 1. Pellet worms and rinse once in 1X M9 salts. 2. For the treatment group, transfer the treatment group of worms to 1X M9 salts with the desired concentration of -amanitin. This exposure is typically done in small volume (0.05mL) in a 1.7mL tube. To set this treatment up, I make a 2X [-amanitin] solution (in 1X M9) and transfer 0.5X of the final exposure volume into the treatment tube. I then transfer worms into the tube in the same volume using a micropipettor. Make an identical control tube without the -amanitin. For a 50L soak, add 25L of -amanitin to 25L of worms in 1X M9. 3. Incubate for desired time (usually 24h) at room temperature with some form of aeration (rocker). **If a timing experiment will be done, also on Day 2, prepare NGM plates with the same -amanitin concentration as in the overnight treatment. Dry the plates briefly and seed with OP50. INCUBATE AT 37C overnight. Day 3: Complete experiment following application-specific protocols.