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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.

  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.

  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.

  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.

  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)

  Bacto tryptone       20.0 g  Bacto yeast extract   5.0 g  NaCl                  0.6 g  KCl                   0.5 g  MgCl2                10 mM (see below)  MgSO4                10 mM (see below)  Glucose              20 mM (see below)  

Note: SOC is identical to SOB, except that it contains 20 mM glucose.

  1. Dissolve tryptone, yeast extract, sodium chloride, and potassium chloride in a final volume of 970 ml distilled H2O. Sterilize by autoclaving.

  2. After autoclaving, allow the solution to cool to 60°C, and add 20 ml of a sterile 1 M glucose stock (see below) to make the media 20 mM with respect to glucose.

  3. Just prior to using, add 10 ml of magnesium stock (see below) to the SOC broth to make the media 20 mM with respect to magnesium.

  2 M Mg2+ stock (100 ml)                      1 M Glucose (100 ml)    20.3 g	MgCl2                                 18.0 g  glucose  24.7 g	MgSO4                                100.0 ml dH2O  

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+.


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Revised: Sunday, September 03, 1995 1:24:52 PM