This protocol describes how to cause RNAi-induced gene silencing via feeding. Use the RNAi section of the intralab site to identify frozen bacterial strains for your gene(s) of interest.
- Get one large ampicillin plate from the deli fridge for every six or so bacterial clones you will use. Divide and label the bottom of each plate, creating a separate section for each clone to grow.
- Take plate(s) and sterile pipette tips to the -80 that has your clones in it (use intralab site RNAi section to identify clones and locations).
- Use separate pipette tips to dig out frozen bacterial clones, streaking each one lightly onto an ampicillin plate.
- Put ampicillin plate(s) in 37C incubator for 16-24 hours.
- Grab about ten colonies from the first clone and put them into a tube with 3 mL LB broth with 100 ug/mL carbenicillin and 50 ug/mL tetracycline (carb stock is 500X, tet stock is 100X). Carb and Tet stocks are on Matt’s level in the general -20 freezer. Repeate for all clones. (Note: Cultures will require 24-48 hours to grow. Other protocols suggest using less antibiotic (50ug/mL carbenicillin and 12.5 ug/Ml tetracycline))
- Put tubes in 37C shaker for 10-14 hours.
- Spot 250 uL of the grown bacteria onto carbenicillin NGM plates. Regular NGM plates also work and may help growth. Tip: Make three plates for each bacterial clone, in case brood sizes are small or one plate fails.
- Let sit for 24 hours or less at room temperature.
- Spot 200 uL of a mix of 0.1 M IPTG and 50 ug/mL carbenicillin in sterile water to induce. 1 M IPTG aliquots are in common -20 freezer with Proteinase K. If not, Matt has dried stuff.
- Let sit right side up at room temperature for at least 24 hours, if not longer.
- Add six or seven L4 worms to each plate (eri-1;lin-15b lines have lower brood sizes, also need to be kept at less than room temperature) and grow in 20C incubator.
- Assay progeny for phenotypes.