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For n transformations into a bacterial strain:
- Culture strain ≈ (1.5×109 × n) cells in liquid medium to the cell quantity in (3 × n) mL saturated LB culture.
e.g. Culture 3 mL E. coli in LB per transformation.., for n · 50 µL transformations.
This is the E. coli quantity in ≈3 mL OD 5 saturated LB culture per 50 µL transformation. - Wash cells twice in ≥⅔ volumes volume 300 mM sucrose, centrifuging room-temp at 16,000×g.
For higher efficiencies or to prevent sparking, wash cells once more in 300 mM sucrose, or in larger volumes.
Keep the sucrose sterile; contaminants can grow in it.
Resuspend cells in 1∕60 vol volume 300 mM sucrose, yielding ~50 µL, 109–1010 cells per transformation.50 µL transformation (2×107 cells/µL)
- Transfer to 1 mM gap cold electroporation cuvette. Tap the cuvette hard twice upon bench to force cells to bottom and release bubbles.
- Wipe cuvette electrodes, and immediately electroporate at 1.8 kV, looking for a time constant ≥3.
- Recovery: Within a few seconds, resuspend the cells in 1 mL rich medium, transfer to culture tube, and recover 37°C shaking for 1–2 hr.
- Or plate without recovery, yielding a few logs lower efficiency.
- Plate on appropriate agar, and incubate at growth-permissive temperature.