---+ 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 μl 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 at -80C. 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 overnight, stationary-phase culture to an OD 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 0.4-0.6. 1 *~30 min before you plan to prepare your cells, set the centrifuge to 4C.* 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 -80C. #TransformingElectroporation ---++ Transforming _E. coli_ Cells by Electroporation This procedure uses the TOP10 Electrocomp™ _E. coli_ cells (but is identical in any other <a target="_blank">[[%ATTACHURL%/New_Gene_Pulser.pdf][standard electromp cell type]]</a>). 1 Thaw the electrocompetent cells on ice. 1 To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA). 1 Mix by gently flicking the tube containing the electrocompetent cell + DNA mixture. Let the mixture sit on ice for 1-10 minutes. 1 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.* 1 Place the cuvette in the pulser and press the "Pulse" button. * For the TOP10 Electrocomp™ _E. coli_ cells, the Ec l setting is fine. 1 After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells. 1 Transfer the mixture to a 1.5 mL microcentrifuge tube. 1 Incubate for ~30-60 minutes at 37°C or other appropriate temperaturein a shaking incubator. * Be sure to place the tube on its side so the transformed cells will grow properly. 1 Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic. 1 Incubate overnight at 37°C or other appropriate temperature. ---+++ 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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