Team:Paris Saclay/preparation

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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= '''Preparation of bacteria super competent cells''' =
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= '''Preparation of supercompetent ''E.coli'' cells''' =
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1. Prepare 2.5 ml of preculture of bacteria for 24 hours.
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1. Prepare the overnight culture  of ''E.coli'' strain DH5alphaZ1 at 37C with shaking at 200-250rpm.
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2. In an Erlenmeyer flask of two liters, inoculate 250 ml of LB with 1 ml of fresh preculture.
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2. In an Erlenmeyer flask of two liters, inoculate 250ml of LB with 2.5ml of overnight culture.
3. The initial optical density of the solution is measured at t0 by spectrometer.
3. The initial optical density of the solution is measured at t0 by spectrometer.
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4. Let bacteria cells grow at 20 °C with agitation at 180 rpm by incubator for 24 h.
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4. Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5
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5. The final optical density of the solution is measured at tfin by spectrometer.
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5. Stop the solution on ice (0 °C) for 10 mins.
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6. Incubate the solution at 0 °C for 10 min.
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6. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).
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7. Centrifuge the solution for 10 min at 4 °C and 6000 rpm.
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7. 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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8. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer, incubate for 10 min at 60 °C.
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8. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).
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9. Centrifuge the solution for 10 min at 4 °C and 6000 rpm.
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9. 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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10. Remove and discard supernatant, resuspend the pellet in 15 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.
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10. Incubate on ice for 10 min.
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11. Incubate the solution at 0 °C for 10 min.
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11. 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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12. Distribute the solution in 150 cold eppendorfs (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.
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<u>Component of Transfo Buffer:</u>
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                        HEPES 10 mmol/L
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                        MnCl2 55 mmol/L
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                        CaCl2 15 mmol/L
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                        KCl 250 mmol/L
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Preparation of 500 ml of Transfo Buffer:
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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, then add 5.44 g MnCl2.
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Sterilize the solution by filtration and store the solution at 4°C.

Latest revision as of 16:04, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Preparation of supercompetent E.coli cells

1. Prepare the overnight culture of E.coli strain DH5alphaZ1 at 37C with shaking at 200-250rpm.

2. In an Erlenmeyer flask of two liters, inoculate 250ml of LB with 2.5ml of overnight culture.

3. The initial optical density of the solution is measured at t0 by spectrometer.

4. Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5

5. Stop the solution on ice (0 °C) for 10 mins.

6. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).

7. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer (see below), incubate on ice for 10 mins.

8. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).

9. Remove and discard 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. 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

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, then add 5.44 g MnCl2. Sterilize the solution by filtration and store the solution at 4°C.



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