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This is directly from a protocol modified by D. Berry from an earlier protocol by Linda Riles and Matt Curtiss.

Make your SLSM: Supplemented Liquid Sporulation Medium.

Make your GNA plates:

All of the ingredients listed below are stored on the chemical shelves in the dark room. If we're out of ingredients, order more from the automated purchasing system- if you don't know how to do this, ask Juniper and then make a wiki page about it.


To make 4 plates worth of GNA Media follow this list taken from the Chemical Storage Room.

Add Stir Bar to Container

5g D-glucose (Reference # 40700008-2)

3g Difco Nutrient Broth (Reference # 234000)

1g Yeast Extract (Reference # 212750)

2g Bacto Agar (Reference # 214010)

Fill container with ddH20 to 100 mL

Mix thoroughly

Autoclave for 15 minutes or Liquid Cycle (learn from someone how to do this!!!). Plates should be freshly made and used within 1-2 days.


Protocol:

1. Grow your cells on YPD plates first. You may be taking these from a tetrad plate or something, which is totally cool. Patch these cells to your first GNA plate and overnight it at 30C.

2. Repatch to another GNA plate for one more day at 30C (you can make both plates at the same time).

3. Transfer to SLSM by resuspending a small amount of your patched (on second GNA plate) cells in 3mL of SLSM.

4. Incubate these on the Tube Roller for 5-7 days at room temp (25C), followed by 3-4 days in the 30C Shaker.

5. This culture may be stored for about a week at 4C, after that the cultures lose viability.