Transformation of Chemically Competent or Electrocompetent E.coli

Gregory Broussard

Revised: 07/20/07

(Please use, change and disseminate freely!)

Transformation of chemically competent E. coli:

1. Thaw cells on ice for about 10 min, one tube of 50µl cells per transformation
2. Add DNA (1-50 ng plasmid) and mix gently by tapping
3. Incubate on ice for 30 min
4. Heat-shock at 42ºC for 45 seconds
5. Immediately place tube on ice for 2 min
6. Add 450 µl TSB and gently mix
7. Incubate at 37ºC for 1 hour (*not any longer)
8. Plate on LB agar containing the correct antibiotics
9. It is recommended to plate 100 µl on one plate and plate the rest on another plate to avoid having a lawn if transformation was very efficient
10. Incubate overnight @ 37ºC

Transformation of electrocompetent E. coli:

1. Thaw cells on ice for about 10 min, one tube of 50 µl cells per transformation
2. Place cuvettes on ice, one per transformation
3. Add DNA (1-50 ng plasmid) and mix gently by tapping
4. Incubate on ice for 10 min
5. Pipet into chilled cuvette
6. Wipe ice/water off the cuvette and tap to remove bubbles
7. Place in electroporator holder
8. Electroporate: Set to 2.5 kV, 200 Ω, 25 ºF
9. Push both buttons simultaneously and hold until beeping sound
10. If it arcs you will hear a popping sound, the cells are likely dead and this transformation must be repeated
11. Add 1 ml TSB to electroporated cells
12. You can add the media directly to the cuvette and then gently pipet it back out into a test tube
13. Incubate at 37 ºC for 1 hour (*not any longer)
14. Plate on LB agar containing the correct antibiotics
15. It is recommended to plate 50 µl on one plate and plate the rest on another plate to avoid having a lawn  (we want single colonies)
16. Incubate overnight @ 37 ºC