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

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(Protocol : Transformation of supercompetent E.coli cells)
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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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# In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.
# In a 1.5 ml eppendorf (3), add 100 μl solution (2) and 900 μl LB.
# 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).
# 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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[https://2014.igem.org/Team:Paris_Saclay/Protocols Back to the protocols]
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Revision as of 12:55, 5 August 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).