A Multiwell Liquid-Based Assay for Screening C. elegans Knockdowns

JoVE Encyclopedia of Experiments

Microbiologie

0 weergaven • 2:35 min • September 26th, 2025

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Take a multi-well plate containing agar media pre-spotted with RNAi bacteria, with or without a gene-specific insert targeting a C. elegans gene.

Seed synchronized L1-stage worms into the wells and incubate them to allow feeding on the RNAi bacteria.

The insert produces interfering RNA that targets specific worm genes, generating a gene-specific knockdown phenotype.

Add buffer to the wells and gently resuspend the worms.

Transfer the suspension to a deep-well plate and allow the worms to settle by gravity.

Remove the supernatant, retaining some buffer.

Wash the worms with buffer, then discard the supernatant, leaving a small amount of buffer.

Next, take a multi-well assay plate containing pathogenic bacteria.

Dispense a defined number of control and knockdown worms into their respective wells to enable host-pathogen interactions. The pathogenic bacteria secrete compounds that induce worm mortality.

Compare worm mortality to assess the effect of gene-specific knockdown on worm survival.

Add one milliliter of S Basal per well of a 24 well plate containing 300 worms per well. Gently agitate the worms by shaking the plate, then transfer worms to an empty, sterile 24 deep well plate.

Allow the worms to settle gravitationally, about five minutes. Aspirate the supernatant, leaving approximately one milliliter per well, Then add 7 milliliters of S Basal to each well. Repeat the wash two more times, and then after the final wash aspirate all but approximately 400 microliters of supernatant.

After pipetting the bacteria into 384 well plates to avoid starvation, using the resampler function of the worm sorter, sort 22 worms into each well of a 384 well plate. Finally, following sorting, add small molecules or other experiment specific materials.

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Laatst bijgewerkt: 15 augustus 2026