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Team:Lyon-INSA-ENS/Realisation/Protocols/CaCl2 trans
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| - | + | This protocol aims at inserting plasmids into NM522 strains | |
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| - | <b>1.</b> | + | <b>1.</b> Monitor the OD600 of a 50mL culture of NM522 cells in LB medium at 37°C. Proceed to the next step when it reaches 0.2-0.4 ( not higher than 0.5 ), which takes approximately 2-3 hours. |
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| - | <b>2.</b> | + | <b>2.</b> Aliquot the culture into 10mL fractions. |
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| - | <b>3.</b> | + | <b>3.</b> Incubate on ice for 15mn |
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| - | <b>4.</b> | + | <b>4.</b> Centrifuge at 4°C, 10mn, 5000rpm |
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| - | <b>5.</b> | + | <b>5.</b> Empty the supernageant |
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| - | <b>6.</b> | + | <b>6.</b> Add 5mL cold and sterile CaCl2 100mM |
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| - | <b>7.</b> | + | <b>7.</b> Incubate 20mn on ice |
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| - | <b>8.</b> | + | <b>8.</b> Centrifuge at 4°C, 5mn, 5000rpm |
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| - | <b>9.</b> | + | <b>9.</b> Empty the supernageant and resuspend ( do not vortex ) in 1mL CaCl2 |
</p> | </p> | ||
Revision as of 13:42, 26 July 2011
CaCl2 chemical transformation
This protocol aims at inserting plasmids into NM522 strains
Procedure
1. Monitor the OD600 of a 50mL culture of NM522 cells in LB medium at 37°C. Proceed to the next step when it reaches 0.2-0.4 ( not higher than 0.5 ), which takes approximately 2-3 hours.
2. Aliquot the culture into 10mL fractions.
3. Incubate on ice for 15mn
4. Centrifuge at 4°C, 10mn, 5000rpm
5. Empty the supernageant
6. Add 5mL cold and sterile CaCl2 100mM
7. Incubate 20mn on ice
8. Centrifuge at 4°C, 5mn, 5000rpm
9. Empty the supernageant and resuspend ( do not vortex ) in 1mL CaCl2
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