Phosphate Limitation Carboy

• Autoclave carboy
• Make 5L stock 10X salts for phosphate limitation:

• Fill 6L carboy/flask with approx. 4L MilliQ H₂O
• Add with constant stirring:

• CaCl₂·2H₂O                            5g
• NaCl                                        5g
• MgSO₄·7 H₂O                       25g
• (NH₄)₂SO₄                          250g
• KCl*                                       50g

• Bring up to 5L with MilliQ H₂O.
• Aliquot into 2L bottles
• Autoclave

To prepare 1 10L carboy:

• Add the following to 1L of MilliQ H₂O in a 1L flask and mix with magnetic stirrer until glucose is completely dissolved:

• 1000x vitamins                        10mL
• 1000x metals                           10mL
• 10 g/L KH₂PO₄ (monobasic)  20mL**
• Dextrose 50g

• Add contents of flask to separate carboy with approx. 7L of MilliQ H₂O, rinse flask into carboy
• Measure out 1L of 10X phosphate limited salts and add to carboy, rinse grad. cylinder into carboy
• Bring up to 10 L with MilliQ H₂O
• Mix for 5 minutes with magnetic stirrer
• Filter sterilize into autoclaved carboy

*NOTE:  this is the new, higher amount of potassium; 7/2005

**NOTE:  Limiting phosphate varies...specify when ordering...default is 20mg/L

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