Preparation of high titer electrocompetent cells

Methods

Preparation of Electrocompetent Cells

Day 1 (end of day; overnight culture)

1. Inoculate a single colony of E. coli from a fresh agar plate into a flask containing 50 mL LB.
2. Incubate overnight at 37 °C with shaking at 250 rpm.

Day 2 (morning start; ~4–5 hours)

Before you begin: Have the following ready and temperature-equilibrated:

• 1 L LB, prewarmed to 37 °C
• 500 mL ultra-pure water, pre-cooled (ice-cold)
• 10% glycerol, ice-cold
• GYT medium, ice-cold

3. Inoculate two separate 2 L flasks each containing 500 mL prewarmed LB with 25 mL of the overnight culture (total: 2 × 500 mL cultures).
4. Incubate at 37 °C with shaking at 300 rpm. Measure OD₆₀₀ every 20 min.

This density is typically reached after ~2.5 hours for DH5α (and often similar for DH10B), but monitor OD₆₀₀ directly.

Critical: Do not allow cultures to exceed OD₆₀₀ = 0.4.

5. When cultures reach OD₆₀₀ = 0.4, rapidly transfer flasks to an ice–water bath for 15–30 min, swirling occasionally for even cooling.

Critical: For maximum transformation efficiency, ensure the temperature of the bacteria does not rise above 4 °C at any stage after chilling.

6. While chilling, place centrifuge bottles in an ice–water bath and pre-cool the centrifuge rotor and chamber to 4 °C.
7. Transfer cultures into ice-cold centrifuge bottles (e.g., 4 × 250 mL).
8. Harvest cells by centrifugation at 1,000 × g (e.g., ~2,500 rpm in a Sorvall SLA 1500 rotor) for 15 min at 4 °C.
9. Decant supernatant and resuspend the pellet in 500 mL ice-cold ultra-pure water (e.g., 2 × 250 mL bottles).
10. Centrifuge at 1,000 × g for 20 min at 4 °C. Decant supernatant and resuspend the pellet in 250 mL ice-cold 10% glycerol (e.g., 1 × 250 mL bottle).

Note: Perform the glycerol resuspension immediately after the spin finishes. Take care when decanting because pellets may lose adherence in 10% glycerol.

12. Centrifuge at 1,000 × g for 20 min at 4 °C. Decant and resuspend the pellet in 10 mL ice-cold 10% glycerol.
    (Optional: transfer to a 50 mL tube and spin in a pre-chilled benchtop centrifuge for later steps.)
13. Centrifuge again at 1,000 × g for 20 min at 4 °C.
14. Carefully decant supernatant. Use a Pasteur pipette attached to a vacuum line to remove any remaining drops of buffer.
15. Resuspend the pellet in 1 mL ice-cold GYT medium.
    This is best done by gentle swirling rather than pipetting or vortexing.
16. Measure OD₆₀₀ of a 1:100 dilution of the cell suspension.
17. Dilute the suspension with ice-cold GYT to 2 × 10¹⁰ to 3 × 10¹⁰ cells/mL.
    Reference conversion: 1.0 OD₆₀₀ ≈ 2.5 × 10⁸ cells/mL.
18. Transfer 40 µL of the suspension into an ice-chilled 0.2 cm-gap electroporation cuvette containing test pUC19 DNA and apply a test pulse to check for arcing.

If arcing occurs: Wash the remaining cell suspension once more with ice-cold GYT medium to further reduce conductivity. If your GYT is ‘off’ you can dilute with ice cold 10% glycerol, incrementally 10% volume adjustments, testing for arcing at each dilution.

20. Dispense 40 µL aliquots into sterile, ice-cold 0.5 mL microfuge tubes.
21. Snap-freeze tubes in liquid nitrogen, then transfer to a –70 °C freezer for storage.