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Finkelstein Lab Wiki

Electrocompetent Bacteria

Contents

Section titled “Contents”
  1. Media
  2. Procedure
  3. Electroporation Protocol
  4. Notes
  5. References

1. Media

Section titled “1. Media”

Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).

2. Procedure

Section titled “2. Procedure”

For two 250 ml cultures.

  1. Get two 1-liter flasks and fill each with 250 ml of SOB.
  2. Inoculate one colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask.
  3. Incubate for 16-18 hours at 37°C and 250 rpm.
  4. Pre-warm the 1-liter flasks to 37°C.
  5. Add two drops of the overnight culture to each of the flasks.
  6. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7.
  7. Turn on centrifuge and cool rotor and centrifuge bottles to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells.
  8. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
  9. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.
  10. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
  11. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells as before.
  12. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
  13. At this point you can electroporate or freeze the cells away. To freeze, Add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen transfer them to a -80°C freezer. The cultures should be good for >6 months.

3. Electroporation Protocol

Section titled “3. Electroporation Protocol”

The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.

  1. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
  2. Place recovery SOC in 37°C water bath.
  3. Pre-warm LB-antibiotic plates at 37°C.
  4. Thaw cells on ice for 10 min or use freshly made cells.
  5. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
  6. Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
  7. Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
  8. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
  9. Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
  10. Rotate in the 37°C incubator for 1 h.
  11. Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:
    1. Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
    2. Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
  12. Incubate overnight at 37°C.

4. Notes

Section titled “4. Notes”

Calculation: If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml) cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)

  • Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice.
  • Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes.
  • Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells. The maximum recommended volume of a DNA solution to be added is 2.5 µl. Addition of a large volume of DNA decreases transformation efficiency.
  • Contaminants such as salts and proteins can lower electroporation efficiency. Ideally, DNA for transformation should be purified and suspended in water or TE. Transformation efficiency is more than 10-fold lower for ligation mixtures than the control pUC19 plasmid due to the presence of ligase and salts. If used directly, ligation reactions should be heat-inactivated at 65°C for 20 min and then diluted 10-fold. For optimal results, spin columns are recommended for clean up of ligation reactions.
  • Electroporation conditions vary with different cuvettes and electroporators. If you are using electroporators not specified in the protocol, you may need to optimize the electroporation conditions. Cuvettes with 1mm gap are recommended (e.g. BTX Model 610/613 and Bio-Rad #165-2089). Higher voltage is required for cuvettes with 2 mm gap.
  • Arcing may occur due to high concentration of salts or air bubbles.
  • It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.
  • Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%.
  • Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation efficiency.

5. References

Section titled “5. References”

Original reference is on the NEB protocols website PDF.