Bologna University/Chemiocompetent cells

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::1. Take some colonies of DH5α cells from fresh streaked plate and inoculate 125 ml of [[Bologna_University/Soc medium | Soc medium]] putting in a 2 l flask.
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::1. Take some colonies of DH5α cells from a fresh streaked plate and inoculate 125 ml of [[Bologna_University/Soc medium | Soc medium]] into a 2 l flask.
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::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).
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::2. Grow the cells overnight at 25 °C (it is advised to grow them slowly in order to have them better synchronized). It takes approximately 20 hours. It is advisable to grow the cells at 25 °C overnight and then to shift them at 37°C. Bacteria are ready for harvesting when 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).
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::3. Pellet the cells at maximum speed at 4 °C for 10 min.
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::3. Spin down the cells (at maximum speed) at 4 °C for 10 min.
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::4. Re-dissolve the pellet in 40 ml of [[Bologna_University/ Transformation buffer | Transformation buffer]].
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::4. Re-dissolve the pellet in 40 ml of [[Bologna_University/Transformation Buffer | Transformation buffer]].
::5. Incubate on ice for 10 min.
::5. Incubate on ice for 10 min.
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::6. Pellet the cells at maximum speed at 4 °C for 10 min.
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::6. Spin down the cells (at maximum speed)at 4 °C for 10 min.
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::7. Re-dissolve the pellet in 10 ml of [[Bologna_University/ Transformation buffer | Transformation buffer]].
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::7. Re-dissolve the pellet in 10 ml of [[Bologna_University/Transformation Buffer | Transformation buffer]].
::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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[[Bologna | Back ]]
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[[Bologna University/Protocols | Back ]]

Latest revision as of 09:21, 25 October 2007

1. Take some colonies of DH5α cells from a fresh streaked plate and inoculate 125 ml of Soc medium into a 2 l flask.
2. Grow the cells overnight at 25 °C (it is advised to grow them slowly in order to have them better synchronized). It takes approximately 20 hours. It is advisable to grow the cells at 25 °C overnight and then to shift them at 37°C. Bacteria are ready for harvesting when 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. Spin down 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. Spin down 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.


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