Eppendorf Multiporator - Electroporation - Manual (Page 17)

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4 Electroporation protocol
4 Electroporation protocol
Electroporation conditions must be optimized for every cell line for which no specific application protocol is available.
The following protocol is a general guideline for the electroporation of eukaryotic cells. To determine the optimal
electroporation conditions for highest transfection efficiency, please refer to Section 3.
1. Ensure that cells are harvested in the exponential growth phase.
2. Dilute the cells in culture medium with 0.5 to 1 % FCS and determine the number of cells and spin the cells down.
3. Resuspend the cells in Eppendorf Electroporation Buffer (at RT or 4 °C) with the determined osmolarity and set a cell
concentration of between 1 x 10 6 and 3 x 10 6 cells/ml, or slightly lower.
Caution: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to
guarantee successful electroporation!
4. Aliquot the cell suspension (400 µl for a cuvette with 2-mm gap width and 800 µl for a cuvette with 4-mm gap width)
in Eppendorf tubes. Add plasmid DNA (final concentration 5 to 20 µg/ml) or proteins (final concentration
10 to 100 µg/ml) and mix.
When performing electroporation at 4 °C, precool the cuvettes on ice.
5. Transfer the cell suspension to electroporation cuvettes. Take care that no air bubbles are formed.
6. Electroporation: (settings on the Multiporator ® )
Mode: Eukaryotic cells
Voltage (U): To enable the optimal voltage to be set on the Multiporator ® , it is advisable to perform a series
of experiments with several pulse voltages.
For adherent cells: between 1 to 5 times the minimum pulse voltage stated in Table 2.
For suspension cells: between 1 to 3 times the minimum pulse voltage stated in Table 2.
Time constant (
τ
): At RT 40 to 100 µs
At 4 °C 15 to 40 µs
Number of pulses (n): 1
7. After pulsing, allow the cell suspension to remain in the cuvette for 5 to10 minutes.
If electroporation was carried out at 4 °C, the cuvettes should be placed on ice for a maximum of 2 minutes after
pulsing and should then be incubated in a water bath for 8 minutes at 37 °C.
8. Carefully remove the cell suspension from the cuvette using a Pasteur pipette and cultivate it in 3 to 5 ml culture
medium in a 60-mm culture dish.
When removing the cell suspension, ensure that the aluminum electrodes are not damaged so that contamination by
cytotoxic aluminum ions is prevented.
Note: After pulsing, the cells should be incubated for 2 to 3 hours at 37 °C before any centrifugation is performed, to
ensure resealing of the membrane.
After the cells have been transferred to the culture medium, they should not be subjected to stress, such as can be
caused by shaking or long periods of transport.
Depending on the cell type and on the plasmid used, transient expression may be detected roughly 24 to 48 hours after
transfection has taken place. In some cases (e.g. primary cells), this may require considerably longer.
4.2 Electroporation procedure
4.3 Follow-up treatment of the cells
4.4 Determination of transfection efficiency in the cells
Multipor_Appli_E_poration_en.fm Seite 41 Montag, 30. Januar 2006 2:17 14