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Methods

John Sulston

Jonathan Hodgkin

DOI: 10.1101/087969307.17.587

Excerpt

I. CULTURE TECHNIQUE
A. Stock Handling
Caenorhabditis elegans is maintained on NGM agar plates carrying a lawn of OP50, a leaky uracil-requiring strain of Escherichia coli (Brenner 1974). For general purposes, 5-cm plates are suitable; 9-cm plates are used for growing larger quantities, and 2-cm plates are useful for experiments with expensive drugs. For growing numerous clones in parallel for screening, 4 × 6-well microtiter plates are convenient; to prevent cross-contamination, the plates must be of a type in which the wells are separated from one another, and to prevent the worms from burrowing, the agar must be overlaid with 2% top agarose.

Streak OP50 on a plate containing rich medium (e.g., TYE); transfer a single colony to rich broth (e.g., PEN) in a screw-capped bottle or culture tube, and leave at room temperature overnight. The resulting suspension keeps for some months at 4°C.

Keep plates at room temperature for at least 3 days before use, so that excess moisture evaporates and those contaminated with fungi and bacteria can be detected and removed. Seed them in batches sufficient for 1–3-day requirements; as a rough guide, a 5-cm plate receives about 1/40 ml of OP50 suspension, streaked with a 1-ml pipette but not touching the wall. At this and subsequent stages, take care not to damage the agar surface, because the nematodes burrow into the breaks and become difficult to score and pick. Allow the lawn to grow overnight at room temperature before nematodes are added. Transfer the nematodes by...


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