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. 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.

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.

1. ~30 min before you plan to prepare your cells, set the centrifuge to 4C.

1. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.

1. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80C.

Transforming E. coli Cells by Electroporation

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.

1. Transfer the mixture to a 1.5 mL microcentrifuge tube.

• • Be sure to place the tube on its side so the transformed cells will grow properly.

1. 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.

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. 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. 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 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.
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.
7. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
8. 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.
9. Resuspend each pellet in approximately 500 μl of 10% glycerol to make a 100x concentration of the initial culture.
10. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80C.

Transforming E. coli Cells by Electroporation

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.

1. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
2. Transfer the mixture to a 1.5 mL microcentrifuge tube.
3. 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.
4. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
5. 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.

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. 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. 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.

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.
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.
7. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
8. 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.
9. Resuspend each pellet in approximately 500 μl of 10% glycerol to make a 100x concentration of the initial culture.
10. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80C.

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.
6. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.
8. 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.
9. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
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.

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. 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. 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.

1. Transfer the cells to 2x 50 ml Falcon conical tubes.
2. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
3. 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.
4. Resuspend each pellet in approximately 500 μl of 10% glycerol to make a 100x concentration of the initial culture.
5. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80C.

Transforming E. coli Cells by Electroporation

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.

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.
2. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
3. Transfer the mixture to a 1.5 mL microcentrifuge tube.
4. 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.
5. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
6. 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.

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.

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.
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 -80C.

Transforming E. coli Cells by Electroporation

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.
6. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.
8. 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.
9. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
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.

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.

• • In general, inoculate to OD₆₀₀ of ~0.05.

1. Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
   • In general, this is an OD₆₀₀ of ~0.6.
2. Transfer the cells to 15 ml Falcon conical tubes.
3. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
4. 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.

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.
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.

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.

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

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.

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.

Electrocompetent _E. coli

Making Electrocompetent _E. coli Cells (small batch)

1. Prepare 10 ml of fresh LB medium in a 50 ml flask for each strain.

1. Transfer the cells to 15 ml Falcon conical tubes.
2. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
3. 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.

Notes

Making Electrocompetent _E. coli Cells (large batch)

1. Prepare 100 ml of fresh LB medium in a 500 ml flask for each strain.

1. Transfer the cells to 2x 50 ml Falcon conical tubes.
2. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
3. 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.

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.

Electrocompetent _E. coli

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.

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.

Electrocompetent _E. coli

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.

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.

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.

Transforming E. coli Cells by Electroporation

This procedure uses the TOP10 Electrocomp™ E. coli cells.

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 Ecl setting is fine.
6. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.
8. Incubate for ~30-120 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.
9. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
10. Incubate overnight at 37°C or other appropriate temperature.

Electrocompetent _E. coli

Making Electocompetent _E. coli Cells

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.

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.

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.

Transforming E. coli Cells by Electroporation

This procedure uses the TOP10 Electrocomp™ E. coli cells.

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 Ecl setting is fine.
6. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.

• • 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.

Electrocompetent _E. coli

Making Electocompetent _E. coli Cells

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.

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.

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.

Transforming E. coli Cells by Electroporation

This procedure uses the TOP10 Electrocomp™ E. coli cells.

1. Thaw the electrocompetent cells on ice.
2. To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA).

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.
2. Place the cuvette in the pulser and press the "Pulse" button.
   • For the TOP10 Electrocomp™ E. coli cells, the Ecl setting is fine.
3. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
4. Transfer the mixture to a 1.5 mL microcentrifuge tube.
5. Incubate for ~30-120 minutes at 37°C in 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.
2. Incubate overnight at 37°C.

Electrocompetent _E. coli

Making Electocompetent _E. coli Cells

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.

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.

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.

Transforming E. coli Cells by Electroporation

This procedure uses the TOP10 Electrocomp™ E. coli cells.

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 minute.
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 Ecl setting is fine.

1. Transfer the mixture to a 1.5 mL microcentrifuge tube.

• • Be sure to place the tube on its side so the transformed cells will grow properly.

1. Clean the cuvettes used.
2. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
3. Incubate overnight at 37°C.

Electrocompetent _E. coli

Making Electocompetent _E. coli Cells

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.

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.

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.

Transforming E. coli Cells by Electroporation

This procedure uses the TOP10 Electrocomp™ E. coli cells.

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 minute.
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 Ecl setting is fine.
6. After electroporation, add 500 μl of SOC to the cuvette to recover the cells.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.
8. Incubate for 60 minutes at 37°C in a shaking incubator.
   • Be sure to place the tube on its side so the transformed cells will grow properly.
9. Clean the cuvettes used.
10. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
11. Incubate overnight at 37°C.

Electrocompetent _E. coli

Making Electocompetent _E. coli Cells

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.

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.

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.

Transforming E. coli Cells by Electroporation

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.

1. Resuspend in approximately 100 µl of 10% glycerol to make a 100x concentration of the initial culture.
2. Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze or proceed directly to electroporation.

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. 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.

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 10% chilled 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.

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 20x the volume of the cell pellet.

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.

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.
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.

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.
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 20x the volume of the cell pellet.

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.

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.
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.

1. Resuspend in 100 µl of 10% glycerol to make a 100x concentration of the initial culture.
2. 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.
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 20x the volume of the cell pellet.

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.

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.
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.
7. 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.
8. Resuspend in 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. 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 20x the volume of the cell pellet.

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.