ElectroCompetent Cells

Preparation of Bacterial Cultures, Tools, and Reagents (DAY 1, Afternoon)

1. In the late afternoon, inoculate 1-5 ml autoclavedLB broth in sterile (for example, 100 x 13 mm) borosilicate glass test tubes with a small aliquot of bacteria (E. coli or Vibrio natriegens).

2. Set the inoculated test tubes in a roller drum housed at 37 °C temperature (warm room or incubator), turn on the roller drum at high speed, and incubate O/N.

3. Prepare a cell spreader in the shape of a "hockey stick" from a glass rod by heating the middle of the rod in the flame of a Bunsen burner
until the glass softens and then direct the bend gently with tweezers or pliers into a 135° angle. Heat the rod once more in the middle point
between the end and the first bend and bend it at a 45° angle inwards (in the opposite direction of the first bend).

4. Prepare LB-agar without antibiotics and pour into Petri dishes in preparation of the electrocompetence protocol, and LB-agar plates
containing the appropriate antibiotic (according to the resistance marker on the vector to be electroporated), and store at 4 °C.

5. Prepare a filter sterilized 2 mM CaCl2 solution for Vibrio natriegens, or autoclave ddH2O for E. coli, and store at 4 °C.

Growth of Electrocompetent Bacteria (DAY 2, Morning)

1. Deliver 100 μl of the O/N bacterial culture onto each LB agar plate, frozen glycerol stocks may also be utilized and directly delivered onto the plate.

2. Dip the hockey stick-shaped cell spreader in 100% EtOH, briefly drain and burn off the remaining EtOH to sterilize it prior to use and spread. Spread the 100 μl O/N bacterial culture evenly with the sterile glass cell spreader making sure not to disrupt (break) the agar surface.

3. Incubate the plate at 37 °C for 4-6 hr, or until a thin lawn of bacterial growth becomes distinguishable. Cells are most competent when actively growing.

Preparation of Electrocompetent Bacterial Cells (DAY 2, Afternoon)

1. Harvest the bacteria with a sterile inoculating loop making sure not to pierce or break the surface of the agar. One 2 mm diameter bacterial mass is sufficient for a single transformation. Typically, this requires scraping the surface of the thin lawn with the inoculating loop two or three times. Several samples (typically between 4-6) may be collected from the same plate.

2. Resuspend the bacterial mass in 1 ml ice-cold 2 mM CaCl2 (for V. cholerae) or sterile ddH2O (for E. coli) and mix well until no clumps are visible, keep on ice.

3. Centrifuge each bacterial suspension for 5 min at 5,000 x g in a refrigerated microcentrifuge set to 4 °C, or in a microcentrifuge stored in a cold room set to 4 °C.

4. Discard the supernatant, resuspend the bacterial pellet in the same volume of ice-cold 2 mM CaCl2 or sterile ddH2O, and repeat the
centrifugation step as done before twice more for a total of three washes.

5. Remove supernatant, resuspend, and then loosen the bacterial pellet thoroughly in 40 μl ice cold 2 mM CaCl2 or sterile ddH2O and keep on ice.

Transformation of Electrocompetent Bacteria (DAY 2, Afternoon)

1. Add up to 1 μg of plasmid DNA (in up to 1 μl water or Tris-EDTA buffer) to the 40 μl bacterial suspension and transfer this mixture into a prechilled, sterile 0.2 cm gap cuvette. The salt concentration in the DNA sample must be lowa, as it will contribute to arcing of the pulse in the next step.

2. Insert the cuvette into the electroporation chamber of the pulse control module, and electroporate at 1.8 kV, 25 μF. The time constant should be ~5.0 msec, and no arcing should occur.

3. Quickly recover the cell suspension by resuspending into 1 ml LB broth and transfer into previously autoclaved borosilicate glass test tube.

4. Allow the cells to recover by incubating under aerated growth conditions (in a roller drum) at 37 °C for 30 min without antibiotic selection.

5. Plate the bacteria onto the previously prepared LB agar plates in the presence of the appropriate selective agent (antibiotic), and incubate at 37 °C O/N. If bacteria are transformed with a high concentration of purified vector (0.1-1 μg supercoiled plasmid), deliver 10 μl of bacterial culture onto the edge of the LB agar plate and with a sterile inoculating loop streak for isolated colonies using the quadrant streak method. If a ligation mixture was transformed, deliver 100 μl of bacterial culture onto each agar plate and evenly spread it with the sterilized glass cell spreader. a If the time constant is short (below 3.5 msec), or pulsing leads