- Streak out cku-80 RNAi strain on LB+Amp+Tet. Also streak out a positive control that produces a visible phenotype (ie. nhr-25).
- Pick single colony into 50 mls of LB+Amp, grow overnight at 37ºC, shaking at 225 rpm.
- Pellet culture at 4000 rpm, 15 minute, remove supernatant and resuspend pellet in 5 mls of LB+Amp. I use this stock for up to 2 weeks.
- Seed 6 cm RNAi plates with 50 µl of the concentrated cku-80 and positive control RNAi bacteria; leave overnight on the bench to induce. I prefer to seed the plates freshly and put worms on the next day.
- Pick four adult pha-1(ts) worms onto the seeded RNAi plates. Incubate at 15ºC. It typically takes 7-8 days to get adults in the F1 generation that one can inject. Confirm RNAi efficacy with the positive control.
- If one expects to be injecting frequently, plate pha-1(ts) on cku-80 RNAi daily during the work week.