Preparing Cells for Electroporation

Cell electroporation is a method that uses an electrical field to increase cell membrane permeability so DNA or other substance can be introduced into the cell.

Reagent Preparation for Cell Electroporation

Table 1. Reagents Required

2× YT medium:	Dissolve 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl in 900 mL of distilled water. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 L with distilled water. Sterilize by autoclaving for 20 min. To prepare as a solid medium, add 1.2% to 1.5% agar.
1 mM HEPES:	0.26 g of HEPES, sodium salt. Dissolve in 900 mL of distilled water. Adjust the pH to 7.0. Adjust the volume to 1 L with distilled water. Sterilize by autoclaving.
10% glycerol in 1 mM HEPES, pH 7.0:	Aseptically add 10 mL of sterile 100% glycerol to 90 mL of sterile 1 mM HEPES, pH 7.0.
10% glycerol in distilled water:	Add 10 mL of 100% glycerol to 90 mL of distilled water. Sterilize by autoclaving.
Isopropanol TE buffer:	10 mM Tris-HCl (pH 8.0), 1 mM EDTA
Phenol:	Redistilled phenol saturated with TE buffer containing 8-hydroxy quinoline
Chloroform/isoamyl alcohol:	Reagent-grade chloroform and isoamyl alcohol, mixed 24:1
Phenol/chloroform:	Equal parts of redistilled phenol and chloroform/isoamyl alcohol (24:1), each prepared as described above
3 M sodium acetate, pH 5.4, aqueous solution Ethanol, 70%, 95%

Procedure

1. Inoculate 10 mL of 2× YT medium with an E. coli host strain from an LB or 2× YT medium plate. Incubate at 37 °C overnight with shaking.
   
   
2. Inoculate 1 L of 2× YT medium with the 10 mL of an overnight culture of host cells. Incubate for 2 to 2.5 h at 37 °C with shaking at 250 rpm until an A₆₀₀ of 0.5 to 0.7 is achieved.
   
   
3. Place the flask on ice for 15 to 30 min.
   
   
4. Spin at 4,000 × g for 20 min at 4 °C.
   
   
5. Decant the supernatant and resuspend the cells in 1 L of ice-cold sterile 1 mM HEPES, pH 7.0.
   
   
6. Spin as described above. Decant the supernatant and resuspend the cells in 500 mL of ice-cold sterile 1 mM HEPES, pH 7.0.
   
   
7. Spin as described above. Decant the supernatant. Wash the cells in 20 mL of sterile 1 mM HEPES, pH 7.0, containing 10% glycerol.
   
   
8. Spin as described above. Decant the supernatant. Resuspend the cells in a total volume of 2 to 3 mL of sterile 10% glycerol in distilled water.
   
   
9. Dispense in 50 to 100 µL aliquots and proceed to the electroporation protocol or freeze on dry ice and store at -70 °C.
   
   
10. Extract the ligated pGEX vector (as well as the uncut vector) once with an equal volume of phenol/chloroform and once with an equal volume of chloroform/isoamyl alcohol.
    
    
11. Remove the aqueous phase and add 1/10 volume of 3 M sodium acetate, pH 5.4 and 2.5 volumes of 95% ethanol.
    
    
12. Place on dry ice for 15 min and then spin in a microcentrifuge for 5 min to pellet the DNA.
    
    
13. Remove the supernatant and wash the pellet with 1 mL of 70% ethanol. Centrifuge for 5 min, discard the supernatant, and dry the pellet.
    
    
14. Resuspend each DNA pellet in 20 µL of sterile distilled water. Alternatively, the DNA can be gel band-purified.

Note: The DNA must be completely free of salt before electroporation.

Electroporation Efficiency

One nanogram of uncut (supercoiled) vector DNA is recommended to be transformed in parallel with insert/pGEX ligations to determine the efficiency of each competent cell preparation. For more information about electroporation protocols, see the instructions for the selected electroporation system.