Eppendorf Multiporator - Electroporation - Manual (Page 8)

32
Calculating the field strength
The critical field strength which is necessary for electropermeation of the membrane can be calculated approximately. To
do so, a rough determination of the diameter (d) of the cell must be made. Based on the determined diameter, the critical
field strength can be calculated using the following formula:
E
c
= V
c
/ (0.75 x d)
E
c
Critical field strength [V / cm]
V
c
Permeation voltage of the membrane [1 V at 20 °C/ 2 V at 4 °C]
d Cell diameter [cm]
The following is an example for a cell with a diameter of 20 µm (2 x 10 -3 cm) at room temperature:
E
c
= 1 V / (0.75 x 2 x10 -3 cm)
E
c
= 667 V/cm
To calculate the voltage which has to be set on the Multiporator ® , it is necessary to multiply the field strength E
C
by the
gap width of the cuvette. In our example, a minimum voltage of 667 V/cm x 0.2 cm = 133 V must be set for a 2-mm
cuvette. For a 4-mm cuvette, twice this value (667 V/cm x 0.4 cm = 267 V) is required.
When electroporation is carried out at 4 °C, the E
C
value is twice as high as the value at room temperature (because
V
C
= 2 V at 4 °C).
At the critical field strength E
C
, pores form on the poles of the cells oriented in the field direction which small molecules
or ions can pass through. It is possible to test "pore formation" immediately using propidium iodide. The red
fluorescence of this dye can be detected when it has been incorporated into the cell and has bound to nucleic acids.
However, for large molecules, such as nucleic acids, the values used must be higher than the critical field strength E
C
.
In the case of suspension cells, the ideal value for introducing plasmid DNA into the cell is normally 1 to 3 times that of
E
C
. For adherent cells, a value 1 to 5 times that of E
C
is necessary to introduce DNA into the cell.
! Eppendorf has step-by-step application protocols for the Multiporator ® for many frequently used cell lines,
which can be found on the Eppendorf homepage at www.eppendorf.com !
Duration of the Soft Pulse
As aforementioned, microsecond pulses are ideal for highly efficient, gentle electroporation. A general rule is that large
cells require a longer time for reversible membrane permeation. With the Multiporator
® , pulses with a time constant
between 5 µs and a maximum of 100 µs are normally used for electroporation. These times are tailored to suit the
Multiporator
® buffer system.
An optimization strategy for new applications with regard to all relevant parameters (e.g. field strengths, pulse lengths)
is described in detail in the following section.
Bibliography
1) Sukhorukov, V.L., Mussauer, H. and Zimmermann, U. (1998) The effect of electrical deformation forces on the
electropermeabilisation of erythrocyte membranes in low- and high conductivity media. J.Membr. Biol. 163, 235-245.
2) Friedrich, U., Stachowicz, N., Simm, A., Fuhr, G., Lucas, K. and Zimmermann, U. (1998) High efficiency electrotrans-
fection with aluminium electrodes using microsecond controlled pulses.
Bioelectrochemistry and Bioenergetics 47, 103-111.
2 Principle of the Multiporator®
2 Principle of the Multiporator®
Multipor_Appli_E_poration_en.fm Seite 32 Montag, 30. Januar 2006 2:17 14