User:Torsten Waldminghaus/Notebook/dnaC2 temperature shift experiment: Difference between revisions
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*Grow 100ml culture in desired medium at 30°C starting the culture with an over night culture 1:1000 | *Grow 100ml culture in desired medium at 30°C starting the culture with an over night culture 1:1000 | ||
*At OD 0.075 shift the culture to 39°C | *At OD 0.075 shift the culture to 39°C | ||
*after 30 min put the flask back to 30°C and add | *after 30 min put the flask back to 30°C and add 4°C medium according to the [[Temperature mixing formula]] to cool the culture directly to 30°C | ||
*Take 5ml sample "0" right before temperature down shift and than at differnt timepoints after downshift (for example at 2, 4, 6, 8, 10, 15, 25 min) | |||
Revision as of 06:20, 12 February 2009
Principle
The dnaC2 mutation in E. coli can be used as tool to synchronize DNA replication. The DnaC protein is needed for initiation of replication. Temperal inactivation via temperature shift of the ts mutant DnaC2 leeds to stop of initiation. Shift back to lower temperature leads to restart of replication.
Protocol
- Grow 100ml culture in desired medium at 30°C starting the culture with an over night culture 1:1000
- At OD 0.075 shift the culture to 39°C
- after 30 min put the flask back to 30°C and add 4°C medium according to the Temperature mixing formula to cool the culture directly to 30°C
- Take 5ml sample "0" right before temperature down shift and than at differnt timepoints after downshift (for example at 2, 4, 6, 8, 10, 15, 25 min)
