Smolke:Protocols/Dropout plates

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(Procedure)
Line 19: Line 19:
#Mix:
#Mix:
#*200 mL NP water (or less, if using alternate sugars)
#*200 mL NP water (or less, if using alternate sugars)
-
#*0.425g YNB
+
#*0.425g YNB (Stored in cold room)
#*1.25g NH<sub>4</sub>SO<sub>4</sub>
#*1.25g NH<sub>4</sub>SO<sub>4</sub>
#*5g Difco agar (''not'' Bacto agar)
#*5g Difco agar (''not'' Bacto agar)

Revision as of 10:06, 12 March 2007

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Contents

Overview

Making selective yeast plates.

Materials

  • Nanopure water
  • Yeast nitrogen base (YNB)
  • Ammonium sulfate
  • Agar
  • Sugars (normally 20% dextrose, alternative 20% raffinose/10% sucrose/20% galactose)
  • Amino acid dropout solution (in 4°C room)

Procedure

N.B. - this protocol makes 12 plates. Adjust volumes as needed.

  1. Mix:
    • 200 mL NP water (or less, if using alternate sugars)
    • 0.425g YNB (Stored in cold room)
    • 1.25g NH4SO4
    • 5g Difco agar (not Bacto agar)
  2. Mix the solution well and autoclave on liquid cycle
  3. After autoclaving, run cold water over the bottle to cool
  4. When bottle is cool enough to touch with the bare hand, add dropout solution and sugar(s)
  5. Swirl to mix, then pour into plates
    • Pour just enough cover the base (shake the plate a little to get an even layer)
    • Briefly flame the top of the plate to eliminate bubbles

Notes

  • Before autoclaving, the mixture will separate into two layers. It should go into solution after autoclaving.
  • If pouring multiple bottles of media, the remaining bottles can stay in the autoclave until needed.
  • I've found that it cuts down on condensation if you pile the plates on top of each other while they dry. -Jkm
  • If you don't want to flame, drop a stirbar into the flask before autoclaving. Mix on the stirplate to add sugar and aa's, stir some more to take care of bubbles. Pour carefully. -tsb

Contact

Win

For another protocol, look here.

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