Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 76)

Section 8
Electroporation of Mammalian Cells
This procedure requires a Gene Pulser Xcell main unit and CE Module.
8.1 Preparation of Electrocompetent Cells
8.1.1 Attached Cells
1. One – two days prior to electroporation, transfer the cells into 75cm 2 flasks with fresh growth medium
so that they will be 50–70% confluent on the day of the experiment. For most cell lines the cell
density will be 2–10 x 10
6 cells / flask; about 1–10 x 10 5 cells are needed per electroporation.
2. Aspirate the media and rinse the flasks using ~12 ml of phosphate buffered saline (PBS).
3. Aspirate the PBS and add ~0.4 ml of trypsin-EDTA; incubate the cells 2–5 min at room temperature.
Tap the flask to detach the cells from the surface.
4. Neutralize the trypsin by adding 10 ml of growth media with serum.
5. Transfer the cells to a 50 ml sterile centrifuge tube; pellet the cells by centrifugation at 400 x g for
5–7 min at room temperature.
6. Aspirate the media, then resuspend the cell pellets in electroporation buffer (Optimem, growth
media without serum, PBS, Hepes-buffered saline, Phosphate-buffered sucrose, or
Hepes-buffered sucrose) at a density of 1–5 x 10
6 cells/ml. Gently pipette the cells to obtain a sin-
gle-cell suspension.
8.1.2 Suspension Cells
1. One – two days prior to electroporation, dilute the cells into fresh growth medium in a 75cm 2 flask
so that they will be in mid-log phase (generally about 0.5–4 x 10 6 cells/ml) on the day of the
experiment. About 1–10 x 10 5 cells are needed per electroporation.
2. Transfer the cells to a 50 ml sterile centrifuge tube; pellet the cells by centrifugation at 400 x g for
5–7 min at room temperature.
3. Aspirate the media, then resuspend the cell pellets in electroporation buffer (Opti-MEM, growth
media without serum, PBS, Hepes-buffered saline, Phosphate-buffered sucrose, or Hepes-buffered
sucrose) at a density of 1–5 x 10
6 cells/ml. Gently pipette the cells to obtain a single-cell suspension.
For all Pre-set Protocols programmed in Gene Pulser Xcell, we recommend that cells be resuspended
in Opti-MEM or in growth media without serum.
8.2 Electroporation
1. Set the desired pulse conditions on the Gene Pulser Xcell. If a Pre-Set Protocol is used (see Table
3.4), open the Mammalian Pre-set Protocols screen as follows: from the Home screen, press 4,
Enter, 3, Enter; use the alpha-numeric keypad or the Up and Down Arrow keys to select the cell
line, then press Enter to open the appropriate Protocol Detail screen.
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