TMP UV integration

TMP/UV Integration protocol.

(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.)