Bacterial mediated RNAi

Contributed by Ian Chin-Sang, Queens University, ON, Canada

Protocol for bacterial mediated RNAi

  • Transform 0.5 ul of RNAi plasmid vector (i.e. L4440, LT61 etc.) into HT115 competent cells.
  • Plate on Amp (50ug/ml) Tet (15 ug/ml) LB plates and grow O/N 37oC.
  • Pick colonies and inoculate 10ml 2XTY Amp (50ug/ml) Tet (15ug/ml) and grow overnight at 37oC (saturated).
  • Once saturated store cultures at 4oC. You may also want to freeze down a sample in 15% glycerol at -80oC. RNAi bacterial cultures should only be kept for no more than 1 month at 4oC. New plasmid transformations or inoculations from frozen stock should be made every month or when needed.
  • Prepare RNAi plates by spreading 65ul (flame hockey stick) to each MYOB worm plate:
  • 5ul Amp (100mg/ml)
  • 5ul IPTG (0.8M)
  • 55ul H2O

If we assume there is about 10ml of MYOB on each plate the final concentrations are roughly 50ug/ml Amp, and 0.4mMIPTG (note we leave out Tet on the plates see below). Make a master mix and scale up for how many plates required. Note the IPTG induction is done on the plate.

  • Seed 100ul (flame hockey stick) of saturated bacteria culture on each plate. Don’t forget to include an empty vector (L4440) control with every RNAi experiment. Also a positive control i.e. fem-1 (LT63 or D9) unc-22 (LT61 or D7) or GFP (L4417 or D11) vectors from the Fire Lab kit. Let seeded plates grow overnight at room temperature and then store at 15oC.
  • Pick 5-6 L 4 animals to each plate. Observe progeny for RNAi phenotypes. It may be necessary to transfer some of the progeny to a new RNAi plate.

This method is based, with permission, on an original protocol available here.