Eppendorf Multiporator - Electroporation - Manual (Page 18)

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If the transfection experiments do not turn out quite as expected, helpful information may be found in the following
troubleshooting guide.
The specific problems listed are caused by a variety of factors which the researcher can easily narrow down by
optimizing specific protocol areas, as described briefly below and outlined in full in Sec. 3 and 4.
Problem Possible cause Solution / comments
Low survival rate Pulse is too strong. Check determination of the cell size (Sec. 4.1.4)
and minimum field strength based on this size
(Table 2). Note that gap width of the cuvette and
temperature during electroporation must also
be taken into consideration.
Pulse is too long. Shorten the pulse length to decrease
permeation of the cell membrane. This can
increase the survival rate of the cells. Note that
the optimal pulse length is affected by the
temperature at which electroporation is carried
out (Sec. 3.6).
Too many pulses are applied. Reduce the number of pulses. Multiple
permeation of the same membrane areas can
lead to irreversible damage to the plasma
membrane.
Conductivity of the electro-
poration buffer is too high. Check the conductivity of your electroporation
buffer using a suitable measuring device.
Buffers with a conductivity of >4mS/cm can
lead to a reduced incorporation rate. Low-
conductivity Eppendorf electroporation buffers
are recommended. The addition of plasmid
DNA dissolved in a buffer solution instead of
distilled water can also increase conductivity.
Cells remained too long in
the electroporation buffer. If overall incubation of the cells in the
electroporation buffer exceeds 30 minutes,
apoptosis maybe inducted in certain cell types.
Shorten the duration of the experiment by
carrying out individual steps more quickly,
by shortening the washing procedure prior to
pulsing, or by cutting the incubation time of the
cells after the pulse. (Attention: An incubation
time of 5 to 10 minutes at room temperature
should be maintained. Then transfer the cells
into culture medium and cultivate at 37 °C).
Before any centrifugation is performed after
pulsing, the cells should be incubated for 2 to
3 hours at 37 °C to ensure resealing of the cell-
membrane.
5 Troubleshooting
5 Troubleshooting
Multipor_Appli_E_poration_en.fm Seite 42 Montag, 30. Januar 2006 2:17 14