---+++ Making Electrocompetent _E. coli_ Cells This procedure makes enough electrocompetent cells for 2-3 transformations. 1 Grow an overnight culture of each strain in LB medium. 1 Prepare 10 ml of fresh LB medium in a 50 ml flask for each strain. 1 Inoculate with 100 ul of the overnight, stationary-phase culture. * In general, inoculate to OD<sub>600</sub> of ~0.05. 1 Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase. * In general, this is an OD<sub>600</sub> of ~0.6. 1 Transfer the cells to 15 ml Falcon conical tubes. 1 Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. 1 Wash by resuspending the cells in 10 ml of 10% chilled glycerol. Repeat centrifugation. Pour off supernatant. Repeat for three wash cycles in 10% glycerol. 1 Resuspend in 100 µl of 10% glycerol to make a 100x concentration of the initial culture. 1 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. 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. 1 Scale up the culture volumes if more electrocompetent cells are needed. The volume of 10% glycerol used for each wash should be at least 20x the volume of the cell pellet. ---+++ References 1 Short Protocols in Molecular Biology, Chapter 1. 1 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.
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Topic revision: r1 - 2008-01-24 - JeffreyBarrick