Team:Paris Saclay/Protocols/Transformation

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='''Protocol : Transformation of supercompetent ''E.coli'' cells'''=
='''Protocol : Transformation of supercompetent ''E.coli'' cells'''=
# Thaw supercompetent cells at room temperature. (almost 100µL for control and 100 µL for one DNA transformation )
# Thaw supercompetent cells at room temperature. (almost 100µL for control and 100 µL for one DNA transformation )
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# Control T-:
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# <u>Control T-</u> :
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In a 1.5 ml microcentrifuge tube (eppendorf), add 100 μl solution of supercompetent cells (0).
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#* In a 1.5 ml microcentrifuge tube (eppendorf), add 100 μl solution of supercompetent cells (0).
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Plasmid DNA pUC18 (or DNA of your choice) :
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# <u>Plasmid DNA pUC18 (or DNA of your choice)</u> :
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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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#* 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).
# Incubate solutions on ice for 30 min.
# Incubate solutions on ice for 30 min.
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# Make a heat shock: incubate bacteria at 42 °C for 1 min, then on ice 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.
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# Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.
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# Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)
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5. Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.
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# In a 1.5 ml eppendorf (2), add 100 μl solution (1) and 900 μl LB.   
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# In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.
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6. Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)
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# 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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* 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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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]]
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Revision as of 14:12, 11 July 2014

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).
  3. 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).
  4. Incubate solutions on ice for 30 min.
  5. Make a heat shock: incubate bacteria at 42 °C for 1 min, then on ice for 2 min.
  6. Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.
  7. Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)
  8. In a 1.5 ml eppendorf (2), add 100 μl solution (1) and 900 μl LB.
  9. In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.
  10. 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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