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).