(r2) Barrick Lab :: ProtocolsElectrocompetentCells

---+++ 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 adding 10 ml of 10% chilled glycerol to each tube, then vortexing vigorously to resuspend the pellet. 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.

Barrick Lab > ProtocolList > ProtocolsElectrocompetentCells

Contributors to this topic

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

Topic revision: r2 - 2008-03-12 - 18:35:23 - Main.JeffreyBarrick