Author: Nagy Lab

Cells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for 1-2 electroporations.

Procedure

  1. Change medium on ES cells 3-4 hours prior to electroporation
  • Gelatinize 10 cm plates, then add 10 ml medium to each.
  • Place them in a 37 0C incubator until they are required.
  • Switch on the electroporation apparatus.
  • Harvest ES cells by trypsinization.
  • Resuspend the cell pellet in ice-cold PBS (1 ml for each 10 cm plate).
  • Determine the cell density (haemocytometer) and dilute with PBS to the required density for electroporation. We regularly electroporate at a relatively high cell density: 7x106 cells/ml (this number varies between different labs).
  • For each electroporation mix together 20-40 microgram (1 µg/µl) DNA (for an approximately 10 kb vector) and 0.8 ml of the ES cell suspension in an electroporation cuvette (BioRad, Cat. No. 165-2088).
  • Set up the electroporation conditions prior to placing the cuvette into the electroporation chamber. We routinely use 250 V, 500 microF for the BioRad GenePulser.
  • Zap the cuvette, then place it on ice for 20 min to 1 hour.
  • Transfer the cells from the cuvette into the prewarmed medium containing dishes. (The contents of one cuvette are routinely seeded into two 10 cm dishes).

post electroporation:

12.Change medium daily.

13.If drug selection is required start this on the second day after electroporation.

14.Continue the selection until colonies become apparent, and grow to a size that is amenable to picking (usually takes 7-10 days).