Janet B. Matsen:Growing Cultures For Crude Extract
Return to Janet's Protocols
- streak fresh plates from freezer stock
- inoculate overnight cultures in rich media for subsequent inoculation
- inoculate into 100 mL of M9 media (or 50 mL of TB) with appropriate antibiotics
- grow to mid-exponential at 37oC, shaking at 225 rpm
- ~0.6 OD600 (says Amanda)
- add inducers if necessary and shake at 18oC & 200 rpm for 36 hours!
Harvest cells by centrifugation
- Split cultures into 2 50 mL tubes.
- Centrifuge for 20 min at 5000 rpm at 4 deg C. (add 10 min if needed)
- Remove supernatant.
- Flash freeze pellet with liquid N2.
- Store at -80 deg C