Procedure

1. Pellet ~5 x 10⁶ cells by centrifugation at 1,500 rpm for 5 min at cold room.
2. Wash the cells twice with ice-cold H-50 buffer.
3. Resuspend the pellet in 100 µl H-50 buffer. Add 1-10 µg of DNA (we haven't tested a minimum amount).
4. Transfer the cell suspension to a cold 0.1 cm electroporation cuvette.
5. Electroporate the cells in the cold at 0.85 kV/25 µF (There is no need for a parallel resistor /time constant is ~0.6 msec) twice with about 5 seconds between pulses.
   Incubate the cuvette on ice for 5 min.
6. Set up 10 cm Petri dishes for selection with 10 ml HL5 per dish. In some cases, I set up multiple for each cuvette so that I can do dilution plating to clone directly.
7. To transfer the cells out of the cuvette, I add a few hundred microliters of HL-5 from the dish to the cuvette, pipette up and down to mix and then withdraw the medium and cells and add to the dish. Repeat once if you want to be sure to get all cells.
8. Add the drug next day.

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Materials

• H-50 buffer (pH 7.0):
  • 20 mM HEPES
  • 50 mM KCl
  • 10 mM NaCl
  • 1 mM MgSO₄
  • 5 mM NaHCO₃
  • 1 mM NaH₂PO₄
  Autoclave and store cold or frozen.


• Antibiotics
  • Blastocidin: 10 µg/ml final concentration from 10 mg/ml stock
  • Hygromycin: 25 µg/ml final concentration
  • G418: 10 µg/ml final concentration

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