---+ 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. 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. Remove promptly and pour off supernatant. 1 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. 1 Resuspend in approximately 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 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. 1 Prepare 100 ml of fresh LB medium in a 500 ml flask for each strain. 1 Inoculate with 1 mL of the overnight, stationary-phase culture. * In general, inoculate to OD<sub>600</sub> of ~0.05. 1 Grow the cells for approximately 3-4 hours, until they reach mid-exponential phase. * In general, this is an OD<sub>600</sub> of ~0.6. 1 Transfer the cells to 2x 50 ml Falcon conical tubes. 1 Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant. 1 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. 1 Resuspend each pellet in approximately 500 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 promptly at -80˚C. When you're ready for electroporation, look at protocol here: [[ProtocolsElectroporation][Electroporation of your Electrocompetent Cells]] ---+++ 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: r12 - 2016-12-15 - SeanLeonard