- 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.
- 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.
- Place the flask on ice for 15 to 30 min.
- Spin at 4,000 × g for 20 min at 4 °C.
- Decant the supernatant and resuspend the cells in 1 L of ice-cold sterile 1 mM HEPES, pH 7.0.
- Spin as described above. Decant the supernatant and resuspend the cells in 500 mL of ice-cold sterile 1 mM HEPES, pH 7.0.
- Spin as described above. Decant the supernatant. Wash the cells in 20 mL of sterile 1 mM HEPES, pH 7.0, containing 10% glycerol.
- 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.
- Dispense in 50 to 100 µL aliquots and proceed to the electroporation protocol or freeze on dry ice and store at -70 °C.
- 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.
- Remove the aqueous phase and add 1/10 volume of 3 M sodium acetate, pH 5.4 and 2.5 volumes of 95% ethanol.
- Place on dry ice for 15 min and then spin in a microcentrifuge for 5 min to pellet the DNA.
- Remove the supernatant and wash the pellet with 1 mL of 70% ethanol. Centrifuge for 5 min, discard the supernatant, and dry the pellet.
- 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.
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.