Pour YPD plates

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Contents

Materials

(Solutions are all available from the media room)

  • 500ml bottle of 2x YEP
  • 200ml bottle of 4% agar -- make sure to sign it out
  • 40% glucose
  • Stack of plates (above the -20 freezers)

Glucose-YEP preparation

  1. Make 2% glucose-YEP mix by adding 50ml of the 40% glucose solution to the bottle of 2x YEP
    • Remember to label and date the bottle after adding the glucose
    • Strictly speaking, this gives you a 2x YEP, 4% glucose solution; this will get diluted appropriately later.
  2. Warm up glucose-YEP mix to ~65 degrees, eg in water bath or incubator

Preparation of LB Agar

  1. Melt in microwave:
    • loosen the cap
    • use 50% power (enter time, press Power, 5, Start)
    • monitor as you melt
    • takes approx. 3 minutes for a 200ml bottle.

Making agar-YEPD mix

  1. Mix agar and glucose-YEP mix by pouring 200 ml of the glucose-YEP mix into the agar bottle -- no need to cool down the agar first, it gets cooled by mixing with the glucose-YEP mixture
    • Since the total volume is now ~400 ml, you now have a 1x YEP, 2% glucose solution, as desired.
  2. Swirl gently to mix, put in water bath for a bit until bubbles disappear

Actual Pouring

  1. Using sterile technique (flame the top of the bottle), pour the YEPD agar into the plates.
    • Cover the base of the plate, and then just a bit more after that.
  2. Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.
  3. Leave plates to dry and cool for a while (overnight even).
  4. Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
  5. Label the bags, using green tape (standard for YEPD)
  6. Put bags in a refrigerator
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