Preparation of Electrocompetent Cells and Electroporation of Plasmid DNA

Preparation of Electrocompetent Cells and Electroporation of Plasmid DNA

1. Inoculate 5-ml L-broth with a single colony of E. coli . Incubate 5 hours to overnight at 37°C on a roller or with moderate shaking.

    

2. Inoculate a volume of L-broth contained in an appropriately sized side-arm flask with one-tenth volume of the culture (i.e. 5 ml of culture to 500 ml L-broth, 2.5 ml of culture in 250 ml L-broth). Grow cells at 37°C with shaking (200-300 rpm) to an OD600 of 0.5 to 0.6.

    

3. Chill the cells in an ice-water bath for 10 to 15 minutes and transfer to a pre-chilled 250-ml centrifuge bottle. Divide the culture if required.

    

   • Cells should be kept at 2°C for all subsequent steps.

      

4. Pellet the cells by centrifugation at 4200 rpm, 2°C for 20 minutes using a Beckman GSA rotor.

    

5. Pour off the supernatant and resuspend the cells in .01 volume ice-cold dH2O (5 ml dH2O for an original culture volume of 500 ml; one-half this amount to each bottle if the culture was divided in step 3. Divide each subsequent volume in half if the culture was divided). Add a volume of ice-cold dH2O equal to the original culture volume and mix well. Centrifuge the cells as in step 4.

    

6. Pour off the supernatant immediately and resuspend the cells in the small amount of fluid remaining in the bottle.

    

   • The pellet may be very loose. Exercise care and pour off the supernatant immediately.

      

7. Add another volume of ice-cold dH2O equal to the original culture volume and mix well. Centrifuge the cells, again as in step 4. Pour off the supernatant immediately and resuspend the cells in the remaining fluid.

    

8. If the cells are to be used directly for electroporation:

    

   1. Place the cell suspension in a pre-chilled, narrow bottom tube of appropriate size and spin for 10 minutes at 4200 rpm, 2°C.

       

   2. Decant the supernatant, and estimate the pellet's volume.

       

   3. Add an equal volume of ice-cold dH2O to resuspend the cells.

       

   4. Keep cells on ice until electroporation is completed.

       

9. If the cells are to be frozen for later use:

    

   1. Place the cell suspension in an appropriately-sized, narrow-bottom tube that has been pre-chilled.

       

   2. Add to the cells an amount of ice-cold 10% glycerol equal to 0.08 of original culture volume (40 ml for a culture of originally 500 ml) and mix well.

       

   3. Spin for 10 minutes at 4200 rpm, 2°C.

       

   4. Decant the supernatant, and estimate the pellet's volume.

       

   5. Add an equal volume of ice-cold 10% glycerol and resuspend the cells (on ice).

       

   6. Aliquot 50-300 µl of the cells to pre-chilled microcentrifuge tubes.

       

   7. Freeze the cells by incubation in a dry ice/ethanol bath. Store at -80°C.

       

   8. When ready to use, thaw the cells on ice.

       

Electroporation of the Cells

1. Set the electroporation apparatus to 2.5 kV, 25 µF. Set the pulse controller to 200 omega.

    

2. Add 1 µl plasmid DNA to tubes containing 40 µl fresh or thawed cells on ice. Mix by swirling with pipette tip.

    

   • The volume of DNA added to the cells should be kept small. Adding DNA up to one-tenth the volume of cells can reduce the efficiency of electroporation 2- to 3-fold.

      

3. Transfer the DNA and cells to a pre-chilled electroporation cuvette (0.2 cm electrode gap) using a narrow pipette tip. Wipe any ice or water from sides of cuvette using a Kimwipe. Place the cuvette into the sample chamber.

    

4. Energize the electroporation apparatus and deliver the pulse by pushing in both charging buttons simultaneously and holding until a short beep is heard. Note the time constant of the pulse and the actual voltage delivered.

    

5. Remove the cuvette from the sample chamber. Immediately add 1 ml SOC medium and transfer the cells to a sterile polypropylene culture tube using a Pasture pipette.

    

   • Failure to immediately add SOC to the electroporated cells can significantly reduce cell viability and decrease transformation efficiency.

      

6. Incubate cultures for 30 to 60 minutes at 37°C on a roller or with moderate shaking to allow for plasmid expression.

    

7. Plate aliquots of the electroporation mixture on L-agar plates supplemented with the appropriate antibiotics. Incubate plates at 37°C.

    

SOC Broth (1 liter)

Dissolve reagents in a final volume Dissolve glucose in 90 ml dH2O. of 100 ml dH2O. Sterilize by filtration Bring to final volume of 100 ml. through a 0.45 µm disposable filter. Sterilize by filtration through a The resulting solution is 2 M with 0.22 µm disposable filter. respect to Mg2+.