Editing ElectroCompetent Cells (section)

== 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.

<br/>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.

<br/>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&nbsp;times. Several samples (typically between 4-6) may be collected from the same plate.

<br/>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.

<br/>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.

<br/>4. Discard the supernatant, resuspend the bacterial pellet in the same volume of ice-cold 2 mM CaCl2 or sterile ddH2O, and repeat the<br/>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.

<br/>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&nbsp;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.

<br/>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&nbsp;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&nbsp;a ligation mixture was transformed, deliver 100 μl of bacterial culture onto each agar plate and evenly spread it with the sterilized glass cell&nbsp;spreader.&nbsp;a If the time constant is short (below 3.5 msec), or pulsing leads