Team:Paris Saclay/Protocols/Preparation of supercompetent Ecoli cells
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(→Preparation of supercompetent E.coli cells) |
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- | + | :'''Component of Transfo Buffer:''' | |
HEPES 10 mmol/L | HEPES 10 mmol/L | ||
MnCl2 55 mmol/L | MnCl2 55 mmol/L | ||
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KOH | KOH | ||
- | Preparation of 500 ml of Transfo Buffer: | + | |
+ | :'''Preparation of 500 ml of Transfo Buffer:''' | ||
* Dissolve 1.19 g HEPES, 1.10 g CaCl<sub>2</sub>, and 9.32g KCl in 500 ml of water, | * Dissolve 1.19 g HEPES, 1.10 g CaCl<sub>2</sub>, and 9.32g KCl in 500 ml of water, | ||
* Adjust the ph at 6.7 with KOH, | * Adjust the ph at 6.7 with KOH, |
Revision as of 15:08, 22 July 2014
Preparation of supercompetent E.coli cells
- Prepare the overnight culture of E.coli strain DH5alphaZ1 at 37C with shaking at 200-250rpm.
- In an Erlenmeyer flask of two liters, inoculate 250ml of LB with 2.5ml of overnight culture.
- The initial optical density of the solution is measured at t0 by spectrometer.
- Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5
- Stop the solution on ice (0 °C) for 10 mins.
- Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).
- Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer (see below), incubate on ice for 10 mins.
- Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).
- Remove and discard supernatant, resuspend the pellet in 20 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.
- Incubate on ice for 10 min.
- Distribute the solution in chilled 1.5ml microcentrifuge tubes (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.
- Component of Transfo Buffer:
HEPES 10 mmol/L MnCl2 55 mmol/L CaCl2 15 mmol/L KCl 250 mmol/L KOH
- Preparation of 500 ml of Transfo Buffer:
- Dissolve 1.19 g HEPES, 1.10 g CaCl2, and 9.32g KCl in 500 ml of water,
- Adjust the ph at 6.7 with KOH,
- Add 5.44 g MnCl2,
- Sterilize the solution by filtration and store the solution at 4°C.