Transformation of Electro-Competent Cells
This requires a pulse generator using high voltages. Be sure you know how to operate it.
- Thaw the electro-competent cells on ice
- Pipet 1 to 3 µl of DNA solution to the cells
- Pipet up and down several times to mix
- Pipet the cell suspension to the pre-cooled cuvet
- Give a pulse of 2500 volts (12.5 kV/cm)
- Check pulse time and voltage.
- Add 700 µl of LB to the electroporated cells
- Transfer the solution to a 1.5 ml eppi
- Incubate for 60-90 minutes at 37 °:C
- Clean the cuvets!
- Plate the cells on a LB plate with antibiotic
- Incubate overnight at 37 °C
1. Thaw the electro-competent cells on ice
2. Pipet 1 to 3 µl of DNA solution to the cells
3. Pipet up and down several times to mix
4. Pipet the cell suspension to the pre-cooled cuvet
Use electroporation cuvet with a 2 mm electrode distance.
5. Give a pulse of 2500 volts (12.5 kV/cm)
6. Check pulse time and voltage.
Pulse time should be above 4.6. If you hear a bang or the pulse time is too low, you had too much salts in the cuvet. Try to use less DNA solution.
7. Add 700 µl of LB to the electroporated cells
8. Transfer the solution to a 1.5 ml eppi
9. Incubate for 60-90 minutes at 37 °:C
Big plasmids should be left to recover for at least 1.5-2 hours.
10. Clean the cuvets!
Wash them in 70% ethanol, then with dextran solution, rinse them well with demiwater, rinse them with 96% ethanol and dry them.
11. Plate the cells on a LB plate with antibiotic
You can split the sample. Plate 50 µl on one plate, spin down the rest 2 min at 8000 rpm, resuspend ca 50 µl you leave in the tube and plate it on a second plate. Like that you will allways have nice colonies.
12. Incubate overnight at 37 °C
- If the pulse times are low, the washing was not sufficient.
References and Comments:
This is a very basic protocol. I got to know this protocol in the Bisseling Lab and did it many times. It works well.
How to cite this page in publications:
This document can be cited like this:
Untergasser A. “Transformation of Electro-Competent Cells” Untergasser's Lab. Winter 2008. (include here the date when you accessed these page).
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