Eppendorf Multiporator - Electroporation - Manual (Page 10)

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The minimum pulse voltage is dependent on the cell diameter following incubation in electroporation buffer as well as on
the temperature and on the gap width of the cuvettes. The values shown can be used to determine the ideal pulse
voltage to be set on the device, as described in Sec. 3.1.
Example
Cell type: Suspension cells
Cell diameter in electroporation medium: ~ 20 µm
Gap width of cuvette: 2 mm
Temperature in the cuvette: Room temperature
Minimum pulse voltage according to diagrams: ~ 130 V
Series of experiments for optimizing pulse voltage: 130 V / 260 V / 390 V
In addition to the field strength, a crucial factor for successful transfection is the pulse length.
The length of the pulse is primarily dependent on the diameter of the cell: the larger the cell, the longer the pulse
necessary for permeation of the membrane.
Empirically, the ideal pulse lengths for electroporation have proved to be 40 to 100 µs at room temperature and
15 to 40 µs at 4 °C. To optimize the duration of the pulse, three different pulse lengths should be selected within the
above-mentioned ranges.
For most cell lines, electroporation is carried out with one pulse.
If one pulse proves to be insufficient, two or more pulses may be used to achieve the desired result, with a 60-second
interval between pulses to allow the cell membrane to regenerate. During multiple pulsing, the Multiporator
® automatically
maintains this interval between each pulse. An intact and resealed cell membrane is a prerequisite for the build-up of the
membrane potential, which is essential for electroporation. In addition, the cells rotate during this regeneration phase
(Brownian movement), which virtually rules out the danger of further "injury" being caused to the same membrane area by
a second pulse.
A precondition for successful electroporation with the Multiporator ® is the special buffer system with low electrical
conductivity.
The best possible transfection results are obtained by using the original buffers from Eppendorf, which have been tested
for sterility as well as for the absence of mycoplasma, endotoxins and pyrogen and which have also undergone a
thorough cytotoxicity test.
However, users may also make up this buffer themselves (see Sec. 7.3).
Ideally, electroporation should be carried out in hypoosmolar buffer, in which the cell absorbs water shortly before the
pulse and then swells up as a result. A number of effects, including a decreased optimal permeation voltage, ensure that
the plasma membrane can be permeated more easily.
3.2 Length of the field pulse
3.3 Number of field pulses
3.4. Adjustment of the electroporation buffer
3 Optimizing the parameters
3 Optimizing the parameters
Multipor_Appli_E_poration_en.fm Seite 34 Montag, 30. Januar 2006 2:17 14