For 1L, mix (add stir bar to bottle):
|4||deionized H2O||1 L|
Place the bottle in the autoclave (121°C for 20 min). Once the autoclave is done (2 hrs), wait until the temperature of the medium drops to 65°C. Put on a hotplate at 65°C and stir. Then add the following, waiting 5 min between each to avoid crystallization:
|5||CaCl2 (1M)||1 mL|
|6||MgSO4 (1M)||1 mL|
|7||KPO4 mix buffer (1M, pH=6, see below)||25 mL|
|8||Cholesterol (stock solution: 5 mg/mL in ethanol)||1 mL|
Stock KPO4 mix buffer: add 108.3 g of KH2PO4 and 35.6 g K2HPO4 to 1 L H2O and autoclave (pH should be at 6).
Connect the pump and pour the plates. Once the distribution is done, pass hot water through the pump tubing to remove the agar.
Let the plates dry for 24 hrs at room temperature and then put the plates in the fridge at 4°C.
Seed NGM plates with OP50
Pick a colony of OP50 bacteria with a sterile toothpick or a pipette tip and put in a Falcon tube containing LB solution. Grow the bacteria overnight on a shaker at 37°C. With a multipette, put 300 uL of solution on each plate. Let the bacteria grow for 2 days and store the plates at 4°C.
Author: Roxane Dervey