C. elegans burrowing choice assay.

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Materials

• PF-127 (Sigma P2443)
• M9
• Water
• Square bioassay dish (e.g. Carolina #741470)
• Large square dish

Advance preparation

1. 2–3 days before the experiment, make a 30% w/v PF-127 gel solution in water. Advance preparation allows the PF-127 to hydrate and dissolve completely.

   • Add 10 mL of water to a 50 mL tube.
   • Weigh out 9 grams of PF-127 solid and add it to the water.
   • Add 11 mL of distilled water to the tube to make a 30% w/v solution.
   • Place the undissolved gel solution at 4°C to dissolve completely over the next 2–3 days.

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   The gel polymerizes at room temperature. Be careful not to take the gel solution out of the fridge too often, be quick when using it, and keep it on ice while out of the fridge.

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2. 5 days in advance of the experiment, pick 5 L4s to each of 5 seeded plates.

Worm-gel preparation

1. By the time you start the experiment, the gel solution should be completely dissolved and the C. elegans should be mostly adults.

2. Wash worms off all 5 plates into a single 1.5 mL tube: add 1 mL of M9 to the first plate, swirl, and transfer to the next plate. Repeat for all 5 plates, then pipette the worms into the tube.

3. Mix the worms by inverting, then allow them to settle for 2 minutes 15 seconds.

4. Repeat the M9 wash twice, then once with water.

5. After the final wash, add up to 1 mL of water to make the titer calculations easier.

6. Count the number of worms in five 5 μL aliquots.

7. Calculate the amount of PF-127 to add to make a 1 worm/μL solution.

8. Set up an ice bath at ~12°C and set an aliquot of the gel solution in a conical tube. Letting the gel warm above 12°C makes it too viscous to work with; colder than 12°C causes greater cold-shock to the worms.

   

   Ice bath at 12°C