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 ul 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. Protocol for Electroporation of your Electrocompetent Cells can be found here.

Notes

1. Keeping the cells cold during all processing steps is recommended. 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. These precautions are not strictly necessary if using the cells for routine cloning.
2. 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)

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 1 mL of the overnight, stationary-phase culture.
   • In general, inoculate to OD₆₀₀ of ~0.05.
4. Grow the cells for approximately 3-4 hours, until they reach mid-exponential phase.
   • In general, this is an OD₆₀₀ of ~0.6.
5. Transfer the cells to 2x 50 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 40 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 each pellet in approximately 500 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 promptly at -80˚C.

When you're ready for electroporation, look at protocol here: Electroporation of your Electrocompetent Cells

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.