Electroporation Protocol

Appendix 3, Extracted from GST Gene Fusion System, GE Healthcare, 2014

Preparation of Cells

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 4000 × 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. Spin 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.

The DNA must be completely free of salt prior to electroporation.

Electroporation

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.