Janet B. Matsen:Growing Cultures For Crude Extract: Difference between revisions
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(New page: Return to Janet's Protocols == growing culture == # streak fresh plates from freezer stock # inoculate overnight cultures for subsequent inoculation # i...) |
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Return to [[ | Return to [[Janet B. Matsen:Protocols|Janet's Protocols]] | ||
== growing culture == | == growing culture == | ||
# streak fresh plates from freezer stock | # streak fresh plates from freezer stock | ||
# inoculate overnight cultures for subsequent inoculation | # inoculate overnight cultures in rich media for subsequent inoculation | ||
# inoculate into 100 mL of M9 media | # 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 | # grow to mid-exponential at 37oC, shaking at 225 rpm | ||
# add inducers | ## ~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 | |||
Latest revision as of 09:52, 13 June 2012
Return to Janet's Protocols
growing culture
- 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
