User:Torsten Waldminghaus/Notebook/dnaC2 temperature shift experiment

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 70 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 [1])
• Mix samples directly with 10ml ice cold Killing Buffer and put on ice (samples should be processed as fast as possible)
• Spin down cells 3 min max. speed at 4°C and take of supernatant

DNA isolation

• resuspend in 300μL [TE] and add 40μL 10%SDS and 3μL 0.5M EDTA
• incubate 5 min at 65°C
• add 750μL isopropanole and mix
• spin at max. speed for 5 min
• resuspend pellet in 500μL TE and add 2μL RNase A (25mg/ml)
• incubate for 30 min at 65°C
• add 2μL protease K (25mg/ml) and incubate at 37°C for 15 min
• phenol extract (2*phenol & 2*chlorophorm)
• precipitate over night with 1ml ethanol and 40μL 3M Na-Acetate
• spin down DNA at 4°C for 15 min, wash in 70% ethanol and resuspend pellet in 50μL A. dest

References

1. Bach T and Skarstad K. Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically. Mol Microbiol. 2004 Mar;51(6):1589-600. DOI:10.1111/j.1365-2958.2003.03943.x |