Lidstrom:M9

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5x M9 Salts

• Using premixed Difco 5x right now

M9 (for 1 L)

1. Add 100 uL 1M CaCl2 to 750 mL autoclaved, warm water (see note)
2. Add 2 mL 1M MgSo4
3. Add 200 mL 5x M9 salts
4. Add carbon source
5. Add any other supplements
6. Add autoclaved water to 1L

• Make sure autoclaved water for diluting the M9, etc. is warm. M9 salts bottle suggests 40-50 deg C. I keep a bottle of autoclaved water for preparing this media in the 65 deg C incubator, so it's ready when I need it.
• Mix between each component especially the CaCl2 and the MgSO4
• Add CaCl2 to the water first.

M9 Agar, 150 mL

1. Add 2.25g of agar to H20 to 100 mL dH2O
2. Autoclave
3. After autoclaving, swirl to mix evenly and "temper" at room temperature until you can place your hand on the flask for 2 second
4. Add 0.3 mL of 1M MgSO4, .015mL of 1M CaCl2 + carbon source of choice
5. Add 30mL 5X M9 salts solution
6. Add H2O to 150 mL

• DO NOT ADD 5x M9 Salts to Agar before autoclaving, otherwise you'll get gross brown agar that nothing will grow on.

• Lab Rat Protocol