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Team:TU Delft/protocols/making competent cells
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1. Cultivate 100 mL LB medium, 37 °C o/n with shaking(140 rpm) | 1. Cultivate 100 mL LB medium, 37 °C o/n with shaking(140 rpm) | ||
| - | 2. Cultivate 50 mL further in | + | 2. Cultivate 50 mL further in 80 mL LB, ~ 2 hours at 37 °C with shaking (140 rpm) until OD601 = 0.4 |
3. Centrifuge 4500 rpm, 6 min, 4 °C | 3. Centrifuge 4500 rpm, 6 min, 4 °C | ||
Revision as of 14:23, 5 July 2010
Making competent cells
Materials:
- bacterial culture
- centrifuge
- 0.1 M MgCl2
- 0.1 M CaCl2
- 80% glycerol
Protocol:
1. Cultivate 100 mL LB medium, 37 °C o/n with shaking(140 rpm)
2. Cultivate 50 mL further in 80 mL LB, ~ 2 hours at 37 °C with shaking (140 rpm) until OD601 = 0.4
3. Centrifuge 4500 rpm, 6 min, 4 °C
4. Resuspend pellet in 80 mL ice-cold 0.1 M MgCl2
5. Centrifuge 4500 rpm, 6 min, 4 °C
6. Resuspend pellet in 50 mL ice-cold 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet, solution should look like milk)
7. Incubate 15 min on ice
8. Centrifuge 4500 rpm, 6 min, 4 °C
9. Resuspend pellet in 40 mL 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet)
10. Incubate 60 min on ice
11. Centrifuge 4500 rpm 10 min 4 °C
12. Add ~1 à 2 mL (depends on the amount of pellet) ice-cold 80% glycerol
13. Divide in parties of 30 µL and quickly put them in the liquid nitrogen
14. Freeze in -80 °C
