Janet B. Matsen:Growing Cultures For Crude Extract: Difference between revisions
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# streak fresh plates from freezer stock | # streak fresh plates from freezer stock | ||
# inoculate overnight cultures for subsequent inoculation | # inoculate overnight cultures for subsequent inoculation | ||
# inoculate into 100 mL of M9 media | # inoculate into 100 mL of M9 media (or 50 mL of TB) | ||
# grow to mid-exponential at 37oC, shaking at 225 rpm | # grow to mid-exponential at 37oC, shaking at 225 rpm | ||
# add inducers (aTc & IPTG) and shake at 18oC & 200 rpm for 36 hours! | # add inducers (aTc & IPTG) and shake at 18oC & 200 rpm for 36 hours! | ||
== storing cells == | == storing cells == | ||
# | # split each between 2 50 mL flasks | ||
# centrifuge for 20 min at 4oC & 5000 rpm (add 10 min if needed) | # centrifuge for 20 min at 4oC & 5000 rpm (add 10 min if needed) | ||
# flash freeze with liquid N2 | # flash freeze with liquid N2 | ||
# store @ -80oC (she says you generally do) in a magic blue box | # store @ -80oC (she says you generally do) in a magic blue box | ||
Revision as of 16:19, 6 June 2012
Return to Janet's Protocols
growing culture
- streak fresh plates from freezer stock
- inoculate overnight cultures for subsequent inoculation
- inoculate into 100 mL of M9 media (or 50 mL of TB)
- grow to mid-exponential at 37oC, shaking at 225 rpm
- add inducers (aTc & IPTG) and shake at 18oC & 200 rpm for 36 hours!
storing cells
- split each between 2 50 mL flasks
- centrifuge for 20 min at 4oC & 5000 rpm (add 10 min if needed)
- flash freeze with liquid N2
- store @ -80oC (she says you generally do) in a magic blue box
