Example protocol for 1L M9 minimum medium cell culture for the N15 or N15/C13 labeled protein

1st day

  1. In the late afternoon, transform 1ul “my” plasmid into 10ul BL21 cells, add 80ul SOC medium, and spread them on a agar plate with Ampicillin, Incubate the plate overnight @ 37°C.

2nd day

  1. In the morning, Pick up a colony and plant it in a 5ml LB with Ampicillin, and shake @ 37°C, 250 rpm to the late afternoon.
  2. Prepare 1L Minimum medium ready (add water and others first, finally add CaCl2, otherwise, precipitation happens!), (just pass through 0.22 um filter, don’t need autoclave) without adding Ampicillin.
  3. In the late afternoon, spin down the 5ml cells, discard the supernatant, and re-suspend the cells with 50ml Minimum medium with Ampicillin, shake O/N at 37°C, 250 rpm.

3rd day

  1. In the morning, transfer the 50 ml culture into 1L minimum medium with Ampicillin (in a 4L big flask), and shake @ 37°C, 250 rpm until O.D. is 0.6 (l = 600nm).
  2. Add IPTG to 0.5mM, and keep shaking overnight at 18C.

4th day

  1. Spin down the cells at 5000 rpm for 10min, and discard the supernatant, and keep the pellets in -80°C.

Hongwei edited 11/19/2014

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