Electrocompetent E. coli

Making Electrocompetent E. coli Cells (small batch)

This procedure makes enough electrocompetent cells for 2-3 transformations.

1. Grow an overnight culture of each strain in LB medium.
2. Prepare 10 ml of fresh LB medium in a 50 ml flask for each strain.
3. Inoculate with 100 μl of the overnight, stationary-phase culture.
   • In general, inoculate to OD₆₀₀ of ~0.05.
4. Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
   • In general, this is an OD₆₀₀ of ~0.6.
5. Transfer the cells to 15 ml Falcon conical tubes.
6. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
7. Wash by adding 10 ml of chilled 10% glycerol to each tube, then vortexing vigorously to resuspend the pellet. Centrifuge for 3.5 minutes. Remove promptly and pour off supernatant. Repeat for at least four wash cycles in 10% glycerol.
8. Resuspend in approximately 100 μl of 10% glycerol to make a 100x concentration of the initial culture.
9. Divide into 30-50 μl aliquots in 0.5 or 1.7 ml tubes. Freeze or proceed directly to electroporation.

Notes

1. Keeping the cells cold during all processing steps is recommended for increased efficiency. Ideally, chill the subculture in an ice water bath for >10 min before centrifuging, use a refrigerated centrifuge or a centrifuge in a cold room, and aliquot the final cell slurry into pre-chilled tubes for storage at -80C. These precautions are not strictly necessary if using the cells for routine cloning.
2. Transformation efficiency depends on both the transformation fraction (what % of the total number of cells are transformed) and the number of cells being transformed. Ensuring that cells are not lost during the wash steps, and the final resuspension results in a 100X concentration is crucial.
3. Scale up the culture volumes if more electrocompetent cells are needed. The volume of 10% glycerol used for each wash should be at least 20× the volume of the cell pellet.

Making Electrocompetent E. coli Cells (large batch, yields 20 50ul aliquots)

1. Grow an overnight culture of each strain in LB medium.
2. Prepare 100 ml of fresh LB medium in a 500 ml flask for each strain.
3. Inoculate with overnight, stationary-phase culture to an OD of ~0.05.
4. Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
   • In general, this is an OD₆₀₀ of 0.4-0.6. Cells have dramatically lower transformation efficiency past exponential phase.
5. ~30 min before you plan to prepare your cells, set the centrifuge to 4C.
6. Transfer the cells to 2x 50 ml Falcon conical tubes. Ensure these tubes are pre-chilled and kept on ice as much as possible.
7. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
8. Resuspend each pellet in chilled 15ml 10% glycerol, and transfer the resuspension to chilled 15ml falcon tubes. The narrow bottom of these tubes forms a more stable pellet, and ensures it is not dislodging while handling.
9. Centrifuge for 5min at 6000 RPM. Discard supernatant, and resuspend the pellet in 15ml 10% glycerol. This resuspension can be accomplished with a serological pipette and/or vortexing. Repeat for a total of four washes. The goal is to remove any traces of media, which contains ions that cause arcing during electroporation.
10. Resuspend each pellet in approximately 500 μl of 10% glycerol to make a 100x concentration of the initial culture. Sometimes, after discarding the supernatant, some liquid may remain behind. Adjust the volume of 10% glycerol added to account for this, otherwise you will end up with diluted cells.
11. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80C.

Transforming E. coli Cells by Electroporation

This procedure uses the TOP10 Electrocomp™ E. coli cells (but is identical in any other standard electromp cell type).

1. Thaw the electrocompetent cells on ice.
2. To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA).
3. Mix by gently flicking the tube containing the electrocompetent cell + DNA mixture. Let the mixture sit on ice for 1-10 minutes.
4. Pipette the mixture into a chilled cuvette, making sure that the mixture is at the bottom of the cuvette by gently tapping the cuvette on a flat surface.
   • Be sure to wipe any condensation off the sides of the cuvette before electroporation.*
5. Place the cuvette in the pulser and press the "Pulse" button.
   • For the TOP10 Electrocomp™ E. coli cells, the Ec l setting is fine.
   • Note electroporation of E. coli is generally carried out at a voltage of 1.8 kV E = 18 kV/cm) when electroporating cells in 0.1 cm cuvettes and at a voltage of 2.5 kV (E = 12.5 kV/cm) when electroporating cells in 0.2 cm cuvettes. These electroporation conditions are pre programmed into the MicroPulser as programs Ec1 (V = 1.8 kV) and Ec2 (V = 2.5 kV) in the bacterial settings menu. Anecdotal evidence suggests that Ec1 setting in 0.1cm cuvettes is ideal for most E. coli strains.
6. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells. Doing this rapidly improves transformation efficiency.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.
8. Incubate for ~30-60 minutes at 37°C or other appropriate temperature in a shaking incubator.
   • Be sure to place the tube on its side so the transformed cells will grow properly.
9. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic. Pre-warming the plates improves plating efficiency.
10. Incubate overnight at 37°C or other appropriate temperature.

References

1. Short Protocols in Molecular Biology, Chapter 1.
2. Datsenko, K.A., and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97: 6640-6645.