TS Cell Electroporation

ES Cell Electroporation    DSM
February 11, 2010
  1. Feed near confluent plate of TSCs with medium 1-2 hr prior to trypsinization
  2. Wash D10 plate with 2 x10 ml PBS.
  3. Trypsinize as for passaging and resuspend in 0.8 ml PBS
  4. Count 10 ul aliquot in hemacytometer & calculate total # cells.
  5. Centrifuge 5x106 cells 1,000 rpm, 5 min, RT.
  6. Wash cell pellet 1 x 2 ml PBS.
  7. Resuspend in 0.8 ml PBS.
  8. Transfer washed cell pellet to 0.4 cm GenePulser electroporation cuvette
  9. Add 4-25 ug linearized DNA.
  10. Electroporate at RT:  0.25 V (KV?), 500 uFD.  Record actual voltage and time constant (~?) after electroporation.
  11. Incubate cells in cuvette 20 min, on ice. Do not add selective agent (e.g. G418, FIAU, hygromycin, etc.) at this time.
  12. Use 2 ml pipet to mix cells in cuvette and plate on 100 mm dish with 10 mL 70% CMF4H
  13. Transfer to CO2 incubator.
  14. Incubate 24 h at 37˚C, 5% CO2.
  15. 24 h after electroporation start suitable drug selection depending on drug resistance gene on plasmid: G418
    (200 µg/mL), puromycin (1 µg/mL), hygromycin (150 µg/mL), etc.
  16. Feed cells with 70% CMF4H plus selectable marker every 48 h.
  17. Pick colonies ~12 d later. Expected yield ~100 drug resistant colonies from 5 x 106 cells in per 100 mm dish.