CloningTransformation of competent E.coli cells with plasmid DNA

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For the preparation of electrocompetent cells follow this protocol.

Note: For incubation on ice, make sure the tubes are standing in an ice-water mix, because without water, the cooling effect of ice is not reproducible due to the air between the ice fragments, especially if you have to incubate for a certain period of time.

Procedure

1. Prechill the cuvettes on ice.
2. Thaw the electrocompetent E. coli cells on ice and transfer 70 µL in the cuvette.
3. Add the DNA solution (ca. 5-10 µL of a ligation reaction or 1 µL of plasmid DNA) and mix gently by shaking the cuvette (do not pipet the cells up and down).
   S settings of the gene pulser: 25 µF , 1.5 kV , 800 Ω
4. Dry the cuvette with a tisue to avoid a bypass.
5. Push the two buttons until the beeping!
6. Add immediately 1 mL SOB (no antibiotics!) and mix by gentle pipetting.
7. Incubate the cuvette 30-45 minutes at 37°C.
8. Plate 10 or 100 µL on LB agar plates containing an antibiotic.
9. Incubate o/n at 37°C.
10. Inoculate 3 mL LB (w/ antibiotic) with a single colony.

Note that increased salt concentration due to high volume of ligation mix can lead to a bypass, thus decreasing the transformation effieciency. Although you can obtain positive transformants in such a case, you should make sure to keep the added volume as low as possible. If necessary you can increase the volume of the cell suspension in order to use more of your ligation mix.

Materials and reagents

• Cuvettes for electroporation (2 mm electrode distance)
• Gene pulser electroporator (BioRad)
• SOB medium
  • 20 g/L bacto-tryptone
  • 5 g/L yeast extract
  • 0.5 g/L mM NaCl 0.186 g/L KCl
  • 10 mM MgCl2*

*Add 10 ml/L 1M sterile MgCl₂ just before use.