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Team:Paris Saclay/Protocols/Transformation of supercompetent Ecoli 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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Revision as of 12:55, 5 August 2014
Protocol : Transformation of supercompetent E.coli cells
- Thaw supercompetent cells at room temperature. (almost 100µL for control and 100 µL for one DNA transformation )
- 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).
- Incubate solutions on ice for 30 min.
- Make a heat shock: incubate bacteria at 42 °C for 1 min, then on ice for 2 min.
- Add 1 ml LB in two eppendorfs, incubate solutions with agitation at 37 °C for 1 h.
- Dilution for the solution (1): ( you are going to plate different dilutions of your pUC18 transformed bacteria)
- In a 1.5 ml eppendorf (2), add 100 μl solution (1) 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).
