Page History

Purpose: A general description of how to make M9 media with

casamino acids, this method makes 200 mL

Materials:

• 5x M9 Salts

• , autoclaved
  • 30 g/L Na₂HPO₄ (anhydrous, "dibasic")
  • 15 g/L KH₂PO₄
  • 2.5 g/L NaCl
  • 5 g/L NH₄Cl₂
• 10% casamino Acids, autoclaved or (preferred) filter-sterilized
• Concentrated Carbon Source, either

• autoclaved or filter-sterilized
• Glycerol
• Glucose
• Etc.
• 1 M MgSO4, Autoclaved
• 1 M CaCl2, Autoclaved
• Antibiotics
• DI Water, Autoclaved
• 250 mL glass bottle

Protocol:

1. Determine amounts to be added
1. 40 mL 5x M9
2. 4 mL 10% CAS amino acids (final concentration 0.2%)
3. 400 uL 1M MgSO4
4. 20 uL 1M CaCl2
5. Appropriate volume of antibiotics
6. Appropriate volume of concentrated carbon source (see notes below)
7. Back solve for the amount of water such that everything adds up to 200 mL
2. Add ingredients into bottle

1. To water, add CaCl2

1. and MgSO4

1. in that order, thoroughly mixing by swirling each time. Then in any order add antibiotics, casamino acids, carbon source, and 5× M9 salts.
1. Note: everything added is sterile, so with proper sterile technique the final solution is sterile and ready to use
2. Color should be clear to slightly yellow
1. Seal bottle with parafilm
2. Store in fridge

Dependencies:

• Requires the making of all solutions listed in the materials section

Known Issues and Workarounds:

• If stored for long periods of time, solutes will fall out of solution, no known workarounds currently
• CaCl2 drops out of solution if not added properly, to avoid this problem add after water and mix before adding other ingredients

Notes:

• Common concentrations of carbon source are:
• Glycerol is kept at 50% (v/v) stock solution (add 1.6 mL to mixture), for 0.4% (v/v)
• Glucose is kept at 20% (w/v) stock solution (add 4 mL to mixture), for 0.4% (w/v)