Bacterial mediated RNAi

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.

Tagged
comments powered by Disqus