C. elegans OP50 Bacterial Lawn Prep Calculator

NGM Plate Prep Timing

NGM plate prep is a multi-day workflow — plan backward from when you need worms.

Make NGM agar (3 g/L NaCl, 17 g/L agar, 2.5 g/L peptone), autoclave, cool to 55 °C, add cholesterol/CaCl₂/MgSO₄/K-phosphate buffer, pour into Petri dishes. Pour at room temperature in a clean hood.

Stack plates upside-down at room temperature for 2–3 days. The surface should be dry and slightly indented but not cracked. Label batches with pour date.

Pick a single colony from a streak plate into LB, shake at 37 °C overnight (~16 h).

Pipette the standard volume onto each plate. Let dry on the bench for 30 min before stacking and incubating.

Lawns are ready for worms 18–24 h after seeding. Plates can be stored at 4 °C for up to 2 weeks.

HT115 RNAi Feeder Substitution

For RNAi by feeding, OP50 is replaced with HT115 carrying a dsRNA expression vector.

HT115 is RNase III–deficient, so dsRNA accumulates in the bacteria instead of being degraded. Worms ingest the bacteria, the dsRNA enters the gut epithelium, and the systemic RNAi machinery silences the target gene throughout the worm.

• LB + 100 µg/mL ampicillin (selects for the dsRNA vector)
• LB + 12.5 µg/mL tetracycline (selects for HT115 itself)
• 1 mM IPTG in LB and on the plate (induces dsRNA expression)
• Otherwise grow exactly like OP50: 37 °C overnight

Use NGM + 1 mM IPTG (instead of plain NGM) for HT115 plates. Seed with 100 µL HT115 culture. The lawn establishes overnight at room temperature. Use within 1 week — HT115 lawns degrade faster than OP50.