Liquid cultures for large scale dauer cultures
From Wikionchus
Liquid cultures can be used to produce dauers in large numbers, and for obtaining dauer pheromone. The method is less prone to bacterial/fungal contamination and is easier to scale up, when compared to the Wet Plate Protocol for the production of "dauer cultures". Also, the fraction of dauers formed is much higher.
Things needed
- S-Medium
- OP50 stock at 20%w/v in S-medium
- Nystatin stock : 50mg/ml in DMSO, Stored at -20C
- Sterptomycin or Kanamycin stock : 20mg/ml in water, Stored at 4C
- Worms : 40 full plates of worms(6cm); OR 16 to 20 full plates of worms(10cm)
Method
In a 2L flask, add the following:
- Wash the worms off the plates using S-medium (50 to 100ml).
- Add S.medium to a final volume of 500ml
- Add 200ul Nystatin
- Add 500ul Streptomycin / Kanamycin
- Add 12.5 ml of 20% OP50 stock
- Incubate in a shaker incubator @ 220rpm, 20C. Higher temperature of 25C can be used for getting more dauers faster.
- Day 3: Check worms, enough dauers should be there. If more dauers are needed, continue the protocol below...
- Day 5: Add 12.5ml of 20% OP50 stock.
- Day 10: Harvest dauers. Do a Dauer purification by Sucrose Floatation and Ficoll precipitation or using modified dauer purification protocol.
Links
- Back to Dauer cultures and Purification page
- Back to Pristionchus pacificus Protocols page
