Team:Paris Saclay/Protocols/Preparation of supercompetent Ecoli cells

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{{Team:Paris_Saclay/protocols_header}}
{{Team:Paris_Saclay/protocols_header}}
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= '''Preparation of supercompetent ''E.coli'' cells''' =
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= '''Preparation of supercompetent ''E. coli'' cells''' =
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# Prepare the overnight culture  of ''E.coli'' strain DH5alphaZ1 at 37C with shaking at 200-250rpm.
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# Prepare an overnight culture  of an ''E. coli'' strain and incubate it at 37°C with shaking at 200-250 rpm.
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# In an Erlenmeyer flask of two liters, inoculate 250ml of LB with 2.5ml of overnight culture.
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# In a 2 liter Erlenmeyer flask, inoculate 250 ml of LB with 2.5 ml of overnight culture.
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# The initial optical density of the solution is measured at t0 by spectrometer.
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# The initial optical density at 600 nm of the solution is measured at t0 with a spectrometer.
# Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5
# Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5
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# Stop the solution on ice (0 °C) for 10 mins.
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# Place the culture on ice (0 °C) for 10 mins.
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# Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).
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# Centrifuge the culture for 10 min at 4 °C at 3000 rpm (2500g).
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# Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer (see below), incubate on ice for 10 mins.
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# Remove and discard the supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer (see below), incubate on ice for 10 mins.
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# Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).
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# Centrifuge the culture for 10 min at 4 °C at 3000 rpm (2500g).
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# Remove and discard supernatant, resuspend the pellet in 20 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.
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# Remove and discard the 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.
# Incubate on ice for 10 min.
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# Distribute the solution in chilled 1.5ml microcentrifuge tubes (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.
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# Aliquot the solution in chilled 1.5 ml microcentrifuge tubes (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.
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:'''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,
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* Adjust the ph at 6.7 with KOH,
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* Adjust the pH at 6.7 with KOH,
* Add 5.44 g MnCl<sub>2</sub>,
* Add 5.44 g MnCl<sub>2</sub>,
* Sterilize the solution by filtration and store the solution at 4°C.
* Sterilize the solution by filtration and store the solution at 4°C.
{{Team:Paris_Saclay/protocols_footer}}
{{Team:Paris_Saclay/protocols_footer}}

Latest revision as of 19:09, 14 August 2014

Preparation of supercompetent E. coli cells

  1. Prepare an overnight culture of an E. coli strain and incubate it at 37°C with shaking at 200-250 rpm.
  2. In a 2 liter Erlenmeyer flask, inoculate 250 ml of LB with 2.5 ml of overnight culture.
  3. The initial optical density at 600 nm of the solution is measured at t0 with a spectrometer.
  4. Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5
  5. Place the culture on ice (0 °C) for 10 mins.
  6. Centrifuge the culture for 10 min at 4 °C at 3000 rpm (2500g).
  7. Remove and discard the supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer (see below), incubate on ice for 10 mins.
  8. Centrifuge the culture for 10 min at 4 °C at 3000 rpm (2500g).
  9. Remove and discard the supernatant, resuspend the pellet in 20 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.
  10. Incubate on ice for 10 min.
  11. Aliquot the solution in chilled 1.5 ml 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.

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