Citrulline Limitation Carboy

• To make 20L stock 10X Nitrogen limited salts:

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

• CaCl₂-2H₂O                 20g
• NaCl 20g
• MgSO₄-7 H₂O            100g
• KH₂PO₄ (monobasic) 200g

o Bring up to 20L with MilliQ H₂O

o Aliquot into 2L media bottles

       o Autoclave

•     To make 500x  citrulline:
  

• Add to ~900 mL Milli-Q water in a 1L glass graduated cylinder and mix well:
• 10g L-citrulline (98%, Sigma C7629 or equivalent)
• Bring to 1L
  
• Autoclave               

For one carboy:

• Autoclave carboy
• Measure ~7L Milli-Q water into a "measuring" carboy
  

• Add the following to ~900ml Milli-Q water in a 1L glass graduated cylinder and mix well:

• 1000x vitamins 10mL
• 1000x metals 10mL
• Glucose                                    50g
• 500x citrulline                         20mL

• When glucose is fully dissolved, add contents of cylinder to "measuring" carboy
• Measure out 1L of 10X Nitrogen limited salts into the cylinder and add to the "measuring" carboy
• Rinse cylinder with Milli-Q water into carboy
• Adjust volume to 10L

• Mix for  ~5min  with magnetic strirrer
• Filter sterilize into autoclaved carboy

for Nitrogen limitation medium see: Dunham, Maitreya. http://genomics.princeton.edu/dunham/MDchemostat.pdf

Modified  by  S. Silverman  10/2006