Ethanol Limitation Carboy: 10

This protocol results in media containing 10 mM Ethanol, 20 mM C

Directions for one carboy:

1. Autoclave carboy

2. Measure ~7L Milli-Q water into a "measuring" carboy

3. Add the following to ~900ml in a 1L graduated cylinder and mix well:

a. 1000x vitamins                       10.0 mL

b. 1000x metals                           10.0 mL

c. Ethanol, 95%                           6.1 mL

4. Add contents of cylinder to "measuring" carboy

5. Measure out 1L of 10X MDS  into the cylinder and  add to the "measuring" carboy

6. Rinse cylinder with Milli-Q water into carboy

7. Adjust volume to 10L

8. Mix for ~5min with magnetic stirrer. Filter sterilize into the autoclaved carboy.

Directions for making 20L stock 10X MDS, in case you need them:

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

• CaCl₂-2H₂O 20g
• NaCl 20g
• MgSO₄-7 H₂O                         100g
• KH₂PO₄ (monobasic) 200g
• (NH₄)₂SO₄ 1000g

Bring up to 20L with MilliQ H₂O

Aliquot into 2L media bottles

Autoclave