Leucine Limitation Carboy for Disome Strains

• Autoclave carboy
• Make 20L stock 10X MDS:

• Fill plastic carboy with approx. 17L MilliQ H₂O
• Add with constant stirring:

• CaCl₂·2H₂O                20g
• NaCl                            20g
• MgSO₄·7 H₂O                        100g
• KH₂PO₄ (monobasic) 200g
• (NH₄)₂SO₄                  1000g

o Bring up to 20L with MilliQ H₂O

o Aliquot into 2L media bottles

o Autoclave

To prepare 1 10L carboy:

• Measure out ~500ml of water and add while stirring
• Adenine sulfate (5mg/mL)      100mL
• Uracil (2mg/mL)                     100mL
• Tryptophan (5mg/mL) 100mL
• Lysine (10mg/mL)                  100mL
• Methionine (5mg/mL)             40mL
• Leucine (10mg/mL)                 15mL
• 1000x vitamins                        10mL
• 1000x metals                           10mL
• Dextrose 50g
• 1g of geneticin
• Add contents of flask to separate carboy with approx. 7L of MilliQ H₂O
• Rinse flask into carboy
• Measure 1L 10X MDS, add and rinse into to carboy
• Bring up to 10 L with MilliQ H₂O
• Mix by stirring for 5 minutes
• Filter sterilize into autoclaved carboy

Dunham, Maitreya. http://genomics.princeton.edu/dunham/MDchemostat.pdf