Team:Paris Saclay/Protocols/Transformation

From 2013.igem.org

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(Protocol : Transformation of bacteria super competent cells)
(Protocol : Transformation of supercompetent E.coli cells)
 
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='''Protocol : Transformation of bacteria super competent cells'''=
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='''Protocol : Transformation of supercompetent ''E.coli'' cells'''=
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1. Thaw bacteria super competent cells at room temperature. (almost 100µL for control and 100 µL for one DNA transformation )
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1. Thaw supercompetent cells at room temperature. (almost 100µL for control and 100 µL for one DNA transformation )
2. Control T-:
2. Control T-:
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In a 1.5 ml microcentrifuge tube (eppendorf), add 100 μl solution of bacteria super competent cells (0).
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In a 1.5 ml microcentrifuge tube (eppendorf), add 100 μl solution of supercompetent cells (0).
Plasmid DNA pUC18 (or DNA of your choice) :
Plasmid DNA pUC18 (or DNA of your choice) :
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In a 1.5 ml eppendorf , add 100 μl solution of bacteria super competent cells, 1 μl (concentration 0.1 ng/μl) of DNA pUC18 (Resistant of Ampicillin)(1).
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In a 1.5 ml eppendorf , add 100 μl solution of supercompetent cells, 1 μl (concentration 0.1 ng/μl) of DNA pUC18 (Resistant of Ampicillin)(1).
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3. Incubate solutions at 0 °C for 30 min.
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3. Incubate solutions on ice for 30 min.
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4. Make a heat shock: incubate bacteria at 42 °C for 1 min, then at 0 °C for 2 min.
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4. Make a heat shock: incubate bacteria at 42 °C for 1 min, then on ice for 2 min.
5. Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.
5. Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.
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6. Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)
6. Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)
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10-1: In a 1.5 ml eppendorf (2), add 100 μl solution (1) and 900 μl LB.
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* 10-1: In a 1.5 ml eppendorf (2), add 100 μl solution (1) and 900 μl LB.
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10-2: In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.
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* 10-2: In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.
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7. Take 100 μl of each solution : the control(0)and the pUC18 transformed bacteria (1),(2),(3) spread them out on four identical environment with ampicillin ( or appropriated antbiotic).
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7. Take 100 μl of each solution : the control(0)and the pUC18 transformed bacteria (1),(2),(3) spread them out on four identical environment with ampicillin ( or appropriated antibiotic).
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[[File:Pstransformation.jpg|center|200px]]
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 18:16, 3 October 2013

Protocol : Transformation of supercompetent E.coli cells

1. Thaw supercompetent cells at room temperature. (almost 100µL for control and 100 µL for one DNA transformation )

2. Control T-: In a 1.5 ml microcentrifuge tube (eppendorf), add 100 μl solution of supercompetent cells (0).

Plasmid DNA pUC18 (or DNA of your choice) : In a 1.5 ml eppendorf , add 100 μl solution of supercompetent cells, 1 μl (concentration 0.1 ng/μl) of DNA pUC18 (Resistant of Ampicillin)(1).

3. Incubate solutions on ice for 30 min.

4. Make a heat shock: incubate bacteria at 42 °C for 1 min, then on ice for 2 min.

5. Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.

6. Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)

  • 10-1: In a 1.5 ml eppendorf (2), add 100 μl solution (1) and 900 μl LB.
  • 10-2: In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.

7. Take 100 μl of each solution : the control(0)and the pUC18 transformed bacteria (1),(2),(3) spread them out on four identical environment with ampicillin ( or appropriated antibiotic).


Pstransformation.jpg