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Fluorescent Protein RNAi Protocol
- Pick an individual colony and inoculate 3mL of LB containing Ampicillin/Carbenicillin (50 µg/ml final) and incubate with shaking at 37℃ overnight. You will need 1 3mL culture to make 3 RNAi plates. Scale as needed.
- While culture is growing, top spread IPTG to a final concentration of 1uM on NGM plates. Typically, 60mm plates contain 7.5mL of NGM. IPTG is made as a 0.5M stock in water. 15 µL of IPTG stock is then diluted to 150 µL (135 µL of water) per plate. 150 µL of diluted IPTG is top spread and allowed to dry for 1hr-overnight.
- Spin down 3 mL of cultured bacteria and resuspend in 150 µL of LB.
- Apply 50 µl of bacteria on the center of each IPTG plate and allow 3hrs-overnight for the bacteria to induce RNAi expression and dry
- On RNAi plates, plate 10-15 egg-laying worms/plate and allow them to lay eggs for 2-6hrs at 25℃.
- After that time, pick off all adult worms and perform experiments on newly hatching worms.
- RNAi effects can be seen as early as the L1 stage.
- Eggs can also be plated directly and RNAi effects are still seen.
- Look for phenotypes associated with protein function.