Team:BCCS-Bristol/Protocols-Transformation using electroporation

Transformation using electroporation

Sterilization of the cuvettes:

1. Open the cuvette and put both (lid and cuvette) into a UV light machine (CL-1000 Ultraviolet Crosslinker from UPV)
2. Give 1200 “energy” (press “start” and wait for the end of the countdown)

Electroporation

1. Prepare 950 μl sterile LB broth in a 1.5 ml tube and put in on ice
2. Put the cuvettes at least 2 mins before adding the bacteria on ice
3. Thaw the electric competent cells (E. coli DH5α) on ice (here 50 μl cells)
4. Add the DNA to the cells (for good working DNA in high concentration like pUC19 with 10 pg/μl use 1 μl; for BioBrick DNA use 5 μl if it was prepared with 10 μl of water)
5. Choose program Ec1 (BIORAD MicroPulser) and change the view to “ms” time
6. Dry the wet cuvette with a paper towel and put it into the machine
7. Give the pulse and notice the time: A pulse between 5-6 ms is a good value
8. Add immediately 950 μl ice cold LB broth and transfer everything back into the tube
9. Incubate for 1 h at 37°C 225 rpm
10. Plate a part of the solution or spin the cells down, remove most of the supernatant (800-900 μl), resuspend them and plate all on a LB plate containing antibiotics
11. Incubate the plates overnight at 37°C