RNAi-voeding in vloeibare cultuur: een methode met hoge doorvoer om genexpressie in C. elegans neer te slaan

- To begin, add carbenicillin and one millimolar IPTG to control and test culture flasks containing S basal media. Spin a tube containing larvae at growth stage one, also called L1 larvae, for a few minutes. Now remove the supernatant and place two microliters of L1s on an agar plate. Place the plate under a dissecting microscope and count the number of larvae.

Add RNAi bacteria carrying a non-specific RNAi plasmid to the control flask and bacteria with gene- targeting RNAi to the test flask. The amount of bacteria added should be proportional to the number of worms.

Let the larvae grow with continuous shaking to ensure proper oxygenation. The larvae feed on the bacteria. Inside the larva, the small interfering RNA binds to the complementary mRNA produced by the targeted gene and inhibits protein expression. Finally, examine the worms for your phenotype of interest.

In the example protocol, we will treat L1 larvae with RNAi targeting the daf-2 gene, in liquid culture.

- To begin treatment, add 207.45 milliliters of S basal media with additional reagents as described in the accompanying text protocol to a 2,800 milliliter Fernbach culture flask. Add a final concentration of 50 micro