Liquid culture, unc-119, co-bombardment protocol

Kevin Kemper and Eric Moss, April, 2004

 

·      All liquid culture is done in 2L non-baffled flasks, ~250mL Complete-S with E. coli, on a shaker at a controlled temperature (See attached detailed protocol on Large-scale synchronized worm preps.)

·      Start with 1-2 10cm plates of unc-119 animals, clean, confluent

·      Seed a 2L flask with ~250mL Complete-S + food (500mL LB culture pellet). We grow at 15°C at this step to reduce dauer formation and to stretch out the ³window of opportunity².

·      Feed regularly using 500-1000mL LB culture pellet at each feeding (added in a reasonably small volume). Donıt allow the worms to starve. Starvation severely curbs fecundity. Worms become voracious as they get older.

·      Grow 1-2 generations until many gravid adults are present in the population.

·      Bleach and seed directly into two 2L flasks with Complete-S and food. (No need to synchronize with starvation arrest.) We grow at 15°C at this step.

·      Grow until population is contains mostly L4s and gravid adults.

·      Float worms on sucrose. You should get about 2mL packed worms.

·      Resuspend 1:1 in cold M9 or water and put onto one chilled 10cm plate without food.

·      Perform bombardment. Recover 30 min.

·      Wash off with M9 or water and distribute into 4-5 2L flasks each with ~250mL Complete-S and food (500mL LB culture pellet).

·      Grow at 25°C.

·      When food runs out, add another dose of food, 500mL LB culture pellet in small volume. Repeat, so that you have fed them for about a week.

·      Let go another week without feeding at 25°C.

·      Spin down worms and collect, keeping contents of each flask separate.

·      Distribute the contents of each flask onto 3 or more seeded 10cm plates.

·      Dauers will crawl out in minutes to hours.

·      We cloned 50-100 dauers per flask and found several giving 100% nonUnc progeny. Others are either hets or arrays. Remember: arrays are common.

·      If doing a co-bombardment, do PCR on stable lines and putative hets to find which ones have both plasmids. Flasks, of course, can be considered independent.