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:
- Determine amounts to be added
- 40 mL 5x M9
- 4 mL 10% CAS amino acids (final concentration 0.2%)
- 400 uL 1M MgSO4
- 20 uL 1M CaCl2
- Appropriate volume of antibiotics
- Appropriate volume of concentrated carbon source (see notes below)
- Back solve for the amount of water such that everything adds up to 200 mL
- Add ingredients into bottle
- To water, add CaCl2 and MgSO4 in that order, thoroughly mixing by swirling each time. Then in any order add antibiotics, casamino acids, carbon source, and 5× M9 salts.
- Note: everything added is sterile, so with proper sterile technique the final solution is sterile and ready to use
- Color should be clear to slightly yellow
- Seal bottle with parafilm
- 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)