Team:TU Delft/protocols/making competent cells

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(New page: === 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 ...)
 
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=== Making competent cells===
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=Making competent cells=
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- bacterial culture  
- bacterial culture  
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- centrifuge
 
- 0.1 M MgCl2
- 0.1 M MgCl2
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- 80% glycerol
- 80% glycerol
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 +
- microcentrifuge
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- spectrophotometer
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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)
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2. Cultivate 50 mL further in 800 mL LB, ~ 2 hours at 37 °C with shaking (140 rpm) until OD601 = 0.4
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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
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5. Centrifuge 4500 rpm, 6 min, 4 °C
5. Centrifuge 4500 rpm, 6 min, 4 °C
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6. Resuspend pellet in 50 mL ice-cold 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet, solution should look like milk)
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6. Resuspend pellet in 50 mL ice-cold 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet)
7. Incubate 15 min on ice
7. Incubate 15 min on ice
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8. Centrifuge 4500 rpm, 6 min, 4 °C
8. Centrifuge 4500 rpm, 6 min, 4 °C
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9. Resuspend pellet in 15 mL 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet)
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9. Resuspend pellet in 40 mL 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet)
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10. Divide in several eppendorf tubes (~ 10)
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11. Incubate 60 min on ice
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10. Incubate 60 min on ice
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12. Centrifuge 14000 rpm 10 min 4 °C
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11. Centrifuge 4500 rpm 10 min 4 °C
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13. Add ~200 µL ice-cold 80% glycerol per eppendorf tube (depends on the amount of pellet)
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12. Add ~1 à 2 mL (depends on the amount of pellet) ice-cold 80% glycerol
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14. Put all the solutions together and divide in parties of 100 µL and quickly put them in the liquid nitrogen
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13. Divide in parties of 30 µL and quickly put them in the liquid nitrogen
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15. Freeze in -80 °C
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14. Freeze in -80 °C

Latest revision as of 09:24, 12 September 2010

Making competent cells

Materials:

- bacterial culture

- 0.1 M MgCl2

- 0.1 M CaCl2

- 80% glycerol

- microcentrifuge

- spectrophotometer


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)

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

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