August/4 August 2009

We prepared a culture of competent E. coli cells according to protocol: 1. An overnight culture of E. coli was inoculated into LB culture solution. 2. Culture was put into 'agitating' incubator set at 37 degrees Celsius and incubated for 1 hr. 3. Optical density (OD) of culture was measured, then the culture was re-incubated. The OD was measured every 30 mins hereafter until it reached/exceeded 0.30. The culture was then equally divided into two centrifugation tubes and left on ice. 4. Culture was ultracentrifuged at 8000 Xg for 5 minutes at 4 degrees Celsius. (Ultracentrifuge must be turned on and temperature adjusted to 4 degrees beforehand) 5. Supernatant was discarded leaving only the pellet, and 0.1M MgCl2 solution added. The centrifugation tubes were then vortexed to dissolve the pellets. 6. Culture was ultracentrifuged again at 8000 Xg for 5 minutes at 4 degrees Celsius. 7. Supernatant was discarded leaving only the pellet, and 0.1M CaCl2 solution added. The centrifugation tubes were then vortexed to dissolve the pellets and incubated on ice for 30 mins. 8. Culture was ultracentrifuged at 8000 Xg for 5 minutes at 4 degrees Celsius. 9. Supernatant was discarded. 750 ul of 1.0M CaCl2 solution and 750 ul of 50% (v/v) glycerol were added to each pellet and the pellets dissolved, giving a total of 1.5 ml culture solution per pellet. 10. The culture solution was then transfered into microtubes (500 ul each). 11. The microtubes containing culture of competent cells were stored at -80 degree Celsius.

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