Janet B. Matsen:Growing Cultures For Crude Extract

Return to Janet's Protocols

growing culture

1. streak fresh plates from freezer stock
2. inoculate overnight cultures in rich media for subsequent inoculation
3. inoculate into 100 mL of M9 media (or 50 mL of TB) with appropriate antibiotics
4. grow to mid-exponential at 37oC, shaking at 225 rpm
   1. ~0.6 OD600 (says Amanda)
5. add inducers if necessary and shake at 18oC & 200 rpm for 36 hours!

Harvest cells by centrifugation

1. Split cultures into 2 50 mL tubes.
2. Centrifuge for 20 min at 5000 rpm at 4 deg C. (add 10 min if needed)
3. Remove supernatant.
4. Flash freeze pellet with liquid N2.
5. Store at -80 deg C