(r12) Barrick Lab :: ProtocolsElectrocompetentCells

---+ 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&times; 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&#730;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.

Barrick Lab > ProtocolList > ProtocolsElectrocompetentCells

Contributors to this topic

JeffreyBarrick, KateElston, DanielDeatherage, AdamHilterbrand, JuliePerreau, SeanLeonard, LindseyWolf, DaciaLeon, AlvaroRodriguez

Topic revision: r12 - 2016-12-15 - 20:49:06 - Main.SeanLeonard

Lab.ProtocolsElectrocompetentCells moved from Lab.ProceduresMakingElectrocompetentCells on 2010-05-07 - 15:02 by Main.JeffreyBarrick -