(r17) ProtocolsElectrocompetentCells < Lab

---+ 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 &mu;l 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 &mu;l of 10% glycerol to make a 100x concentration of the initial culture.
   1 Divide into 30-50 &mu;l 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 &gt;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 20Ã&mdash; 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 &mu;l 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&trade; _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 &mu;l of DNA (&lt;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&trade; _E. coli_ cells, the Ec l setting is fine.
   1 After electroporation, add 500-1000 &mu;l 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 &mu;l) 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.
Barrick Lab > ProtocolList > ProtocolsElectrocompetentCells
Topic revision: r17 - 2018-12-10 - 20:45:49 - Main.KateElston
Lab.ProtocolsElectrocompetentCells moved from Lab.ProceduresMakingElectrocompetentCells on 2010-05-07 - 15:02 by Main.JeffreyBarrick -