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TMP/UV Integration protocol

(from Scott Clark, modified by Kimble lab)

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

Materials

  • 3 mg/mL TMP (trimethylpsoralen) dissolved in DMSO
    NOTE: University of Wisconsin Madison safety protocol for TMP is to keep the 10 cm plates the TMP was aliquoted onto in a plastic bag and schedule Safety pick up. Tips and tubes can go in autoclave trash.
  • UV Stratalinker 1800
  • M9
  • 1.5 mL microcentrifuge tubes
  • Tin foil
  • 10 cm unseeded NGM plates
  • OP50 in liquid culture

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 due to death of worms). Add 20 µL 3 mg/mL TMP (trimethylpsoralen in DMSO) to each 1.5 mL microcentrifuge tube in the dark as TMP is light sensitive.

  2. Wrap 1.5 mL microcentrifuge tubes in foil and incubate at RT for 15 min.

  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 350f µJ (x100) long wave UV in the Stratalinker 1800 without the lid.

    Stratalinker 1800 operation instructions: - set plates on paper towel in the Stratalinker 1800 (located in Lyric's lab 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 re-suspend. Add 1 mL of this concentrated E. coli 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 (pick 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 1-25 6 cm plate and incubate at 20°C.

  8. Day 4 Transfer the same original parent worm onto a new numbered 1-25 6 cm plate and incubate at 20°C.

  9. Day 5 Pick 100-120 6 cm plates with 2 transgenic worms per numbered 1-25 6 cm plate from the 75 plates picked over the last 3 days. There will be some death of worms so you may not have 75 plates anymore, just pick from what you have left.

  10. Day 8 From each of the 100-120 plates from step 9, pick 2 transgenic worms to individual 6 cm plates (you should end up with about 250 plates).

  11. Day 11 Score each plate for 100% integration of the transgenic marker.

  12. Day 12 Score each plate for 100% integration of the transgenic marker.

Backcrossing Integrated lines to remove mutations

  1. Select 4 transgenic, integrated L4 hermaphrodites and place the worms on one 6 cm plate and let them crawl around for 5 minutes.

  2. Add 7-10 L4 male worms from the nonmutagenized, nontransgenic parental strain to the plate with the hermaphrodites.

    Example: A line expressing GFP in the pharynx was generated by microinjection into C. elegans strain N2 and taken through the UV TMP integration protocol. The line with GFP in the pharynx would be the transgenic, integrated line and N2 would be the nonmutagenized, nontransgenic parental strain.

  3. Incubate at 20°C for 3.5 days.

  4. On a new 6 cm plate, select 4 nonmutagenized, nontransgenic parental strain L4 hermaphrodites and let them crawl around for 5 min.

  5. Add 7-10 transgenic, integrated L4 male worms from the plate from step 3.

  6. Repeat steps 3-5 for 5-6 additional generations. If you cannot find transgenic males, you may have an insertion on the male chromosome. You can then take transgenic hermaphrodites and mate to males from the nonmutagenized, nontransgenic parental strain.

  7. Pick 12 single bright transgenic hermaphrodites (one per plate) from final backcross plate.

  8. Incubate at 20°C for 3.5 days.

  9. Check for 100% integration from one of the 12 plates. This will be the new integrated line.

  10. Freeze down the new integrated strain.