TMP/UV integration protocol, from Scott Clark, modified by the Kimble lab.

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This is a multi-day protocol. To avoid weekends, day 1 should be a Monday.

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Materials

• 3 mg/mL TMP (trimethylpsoralen) dissolved in DMSO
• UV Stratalinker 1800
• M9
• 1.5 mL microcentrifuge tubes
• Tin foil
• 10 cm unseeded NGM plates
• OP50 in liquid culture

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UW–Madison safety protocol for TMP: keep the 10 cm plates the TMP was aliquoted onto in a plastic bag and schedule a Safety pick-up. Tips and tubes can go in autoclave trash.

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Mutagenesis and integration

1. Day 1. In 380 μL M9, add 40 L4 worms to be integrated in a 1.5 mL microcentrifuge tube. Do 2 tubes, to allow for worm death. Add 20 μL of 3 mg/mL TMP to each tube in the dark, as TMP is light sensitive.
2. Wrap the tubes in foil and incubate at room temperature for 15 minutes.
3. Transfer worms to two 10 cm unseeded plates, one tube per plate, in the dark. Swirl the liquid around and cover the plates with foil.
4. Expose the worms to 350 μJ (×100) long wave UV in the Stratalinker 1800, without the lid.
   • Set plates on a paper towel in the Stratalinker 1800 (room 327).
   • Turn on.
   • Hit "Energy".
   • Choose 350 μJ.
   • Push start.
5. Spin down about 10 mL E. coli OP50, remove all but 1 mL of liquid, and resuspend. Add 1 mL of this concentrated OP50 to the plates, cover in foil, and incubate at 15°C overnight.
6. Day 2. Pick 1 surviving transgenic worm per 6 cm plate, to about 25 plates. Number the plates 1–25 to keep track of the original parent. Incubate at 20°C.
7. Day 3. Transfer the same original parent worm onto a new numbered 6 cm plate and incubate at 20°C.
8. Day 4. Transfer the same original parent worm onto a new numbered 6 cm plate and incubate at 20°C.