"

From 2014.igem.org

---

Revision as of 10:00, 12 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).

Back to the calendar