Appendix 4: Non-Inducing and Autoinduction Media

Non-Inducing Media for Starter Cultures

When inoculating auto-induction media with cells, saturated rich media starters (i.e., saturated cells in 2XYT broth) can be used.  However, saturated starters grown in non-inducing media can also be used, and also are stable for a much longer period of time when stored at 4 °C (for up to, or even over, a week).  To make 1 liter of non-inducing media, follow the directions below.

 

Non-inducing media for starters (1L)

Aspartate (5%, pH 7.5)                    50 mL

25X Mineral salts                             40 mL

Glucose (40%)                                  12.5 mL

MgSO4 (1 M)                                    2 mL

Trace metals (5000x)                     200 mL

18 AA mix (25X) (@ 4°C)              40 mL  (for faster starter growth)

 

Add appropriate antibiotics (ampicillin to final concentration of 100 mg/mL and tetracycline to 25 mg/mL).

Add sterile water to final volume of 1 L.

Approximately 4-5 mL of non-inducing media is sufficient for a starter of a particular cell culture.  Inoculate this volume of non-inducing starter with 40-50 mL of saturated 2XYT or other rich media starter.

 

Autoinduction Media for Expressions

To make 100 mL of autoinduction media (AIM), follow the directions below.

Scale the make amount you need for your experiment based on the following amounts per 100 mL.  Prepare fresh and do not plan on storing extra AIM.

Aspartate (5%, pH 7.5)                       5 mL

Glycerol (10%)                                     5 mL

25x Mineral Salts                                4 mL

Glucose (40%)                                     0.125 mL

MgSO4 (1 M)                                       0.2 mL

Arabinose (20%)                                 0.25 mL

Trace metals (5000x)                         20 mL

18 AA mix (25x) stored @ 4°C          4 mL

 

Add appropriate antibiotics (ampicillin to final concentration of 100 mg/mL and tetracycline to 25 mg/mL).

Add sterile water to a final volume of 100 mL.

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