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  • Protocol I: Yeast Agar Medium with 5 mM acetamide solution (500 ml)

    Protocol

    1. Mix in an autoclavable bottle:
      a. 1M Tris-HCI Buffer Stock Solution (see Protocol II) 15 ml
      b. YCB Medium Powder (supplied with kit) 5.85 g
      c. Bacto agar (Becton Dickinson #214050) 10 g
      d. Bring volume up to 495 ml with dH2O
      e. Autoclave 20 minutes at 121°C.
      Let cool to approximately 60°C.
    2. Aseptically add 5 ml of sterile 100X acetamide solution.(supplied with kit)
    3. Dispense into sterile disposable Petri dishes; Close plates and let sit at room temperature until solid, then invert and let sit for 12–18 hours to dry prior to use. 

      Yeast carbon base (YCB) medium contains glucose and all nutrients needed to sustain growth of K. lactis GG799 Competent Cells except a simple nitrogen source. Cells can utilize acetamide as a source of nitrogen only after it is broken down to ammonia by acetamidase (the product of the amdS gene present in pKLAC2). Acetamide should not be autoclaved.