Dropout plates for yeast

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Revision as of 17:30, 8 May 2007 by Reshma P. Shetty (Talk | contribs)
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Contents

Materials

(Solutions are all available from the media room)

  • 200ml bottle of 2x SD
  • 200ml bottle of 4% agar -- make sure to sign it out
  • 40% glucose
  • CSM minus the relevant marker(s), as powder
  • Stack of plates (above the -20 freezers)

Yeast growth medium generally consists of three parts: a nitrogen base (supplied by SD), a sugar source (supplied by glucose) and necessary amino acids (supplied by CSM). These three components are what you're going to mix together and then add to agar to make plates.

Making amino acid dropout (CSM) solution

Generally easiest to make a 10x master solution that you can dilute back when you're pouring plates. Let's assume you're going to make 200ml of a 10x master solution:

  1. Grab a 500 ml conical flask
  2. Figure out how much of your dropout powder you're going to need and weigh it out
    • For example, the CSM - TRP mix we have is 0.74g/L, so for a 200ml of a 10x solution, you'll need 10*0.74/5 = 1.48g.
  3. Dump CSM into conical flask and fill it up to 200ml mark with deionized water (from white tap above the sink)
  4. The CSM will need a bit of help dissolving, so put in a stir bar (rinse it off first with dI water) and put it on a stirrer for 1-2 hours (speed around 6-7, heat 3-4).

Once the CSM has gone into solution, you'll need to filter-sterilize it:

  1. Get a 200ml orange screwtop bottle and a vacuum filter
  2. Flame the bottle & top, remove the filter from its packaging and screw it onto the bottle. The filter is sterile, so try not to touch it to anything else
  3. Attach the adapter to the filter and the vacuum nozzle to the adapter (vacuum should be off)
  4. Pour the CSM solution into the top of the filter
  5. Turn on vacuum and allow the solution to get sucked through the filter and then turn off the vacuum
  6. Unscrew the filter, flame the bottle and top again, recap it and label it.
  • Alternatively, see here to make DO stocks from individual amino acids.

Mixing growth medium

  1. Add 20ml of 40% glucose and 40ml of 10x CSM solution to 200 ml of 2x SD
  2. Heat up mixture to 65 degrees in water bath

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. 3-5 minutes for a 200ml bottle.

Making agar-growth medium mix

  1. Mix agar and growith medium pouring the growth medium into the agar bottle -- no need to cool down the agar first, it gets cooled by mixing with the growth medium
    • Since the total volume is now ~400 ml, you now have a 1x everything, 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 agar mixture 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 correct color tape for the dropout medium
  6. Put bags in a refrigerator
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