Bologna University/Chemiocompetent cells
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::8. Add 700 μl of DMSO. | ::8. Add 700 μl of DMSO. | ||
::9. Aliquot (200 μl) and freeze at -80°C. | ::9. Aliquot (200 μl) and freeze at -80°C. | ||
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Revision as of 10:38, 3 August 2007
- 1. Take some colonies of DH5α cells from fresh streaked plate and inoculate 125 ml of Soc medium putting in a 2 l flask.
- 2. Grow the cells overnight at 25 °C (it is better to grow them slowly in order to be better synchronized). It takes approximately 20 hours. It is advisable to grow the cells at 25 °C overnight at 25 °C and then shift them at 37°C. Bacteria are ready for harvest when the OD600 is between 0.37 and 0.4. Higher OD will lead to less competent cells (it is important to harvest the bacteria when they are still in the logarithmic phase of growth).
- 3. Pellet the cells at maximum speed at 4 °C for 10 min.
- 4. Re-dissolve the pellet in 40 ml of Transformation buffer.
- 5. Incubate on ice for 10 min.
- 6. Pellet the cells at maximum speed at 4 °C for 10 min.
- 7. Re-dissolve the pellet in 10 ml of Transformation buffer.
- 8. Add 700 μl of DMSO.
- 9. Aliquot (200 μl) and freeze at -80°C.
