TMP UV Integration¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
TMP UV Integration (from Scott Clark)
- Recover L4 array-containing animals in 200ml of M9.
- Add 20ml of 1mg/ml TMP (Trimethyl Psoralen in DMSO) to 380ml M9, resulting in final concentration 50mg/ml.
Note, Although freshly made TMP solution is probably best, I have used TMP sol’n that has been stored wrapped in foil at -20C as well. It appears OK for a few weeks or more
- Add worms to TMP sol’n.
- Let worms sit for 15 minutes, covered at RT.
- Transfer to unseeded large plate in dark.
- Expose worms to 350mJ(x100) long wave UV in Stratalinker 1800 (without lid).
- Add some concentrated OP50, and maintain worms covered for 5 hours at RT or O/N at 15C.
- Pick 2-3 L4 animals/plate for 8 plates, as P0s. (way extra, but convenient)
- Transfer P0s to new plates every 24 hours for 2-3 days.
- Pick 100-120 F1, one per plate.
- Pick 2 F2 from each F1 plate. one/plate.
As some F1s will be sterile, I will pick extra F2s from some plates to get 200-250 total F2 animals
- Score for 100% non-Muv, Rol, or gfp or whatever.
- Typically recover 1-3 integrants/100 F1. (compared with 1-3 integrants/3-400 F1s by X-ray mutagenesis.)
This method is based, with permission, on an original protocol available here.