Eppendorf Multiporator - Electroporation - Manual (Page 19)

43
Gene product has
a toxic effect on the cell. The gene product itself or a high expression rate
of the gene product may have a toxic effect on
the cells. The optimal plasmid concentration
should be tested individually for each cell type
and each plasmid.
After pulsing, cells are incubated
too long at a low temperature
(4 °C). An excessive incubation period on ice can lead
to cell death. The incubation period on ice
should not exceed two minutes.
The cells are damaged during
the harvesting procedure. Using trypsin with an excessively high concen-
tration or with an insufficient purity level during
the harvesting of the cells may increase the
mortality rate. Dispase or trypsin HPLC-grade
(Sec. 4.1.3.1) is recommended.
Osmolarity of the electro-
poration buffer is too low. Low osmolarity of the electroporation buffer
may cause sensitive cells to swell to such an
extent that they burst during electroporation.
This effect can be tested by a viability staining
of the cells 2–3 hours after electroporation
(Sec. 4.1.3.2). Increase the amount of
isoosmolar electroporation buffer to raise the
osmolarity of the buffer and repeat the viability
test.
Cells are stressed. At the time of electroporation, the cells should
have been in culture for several cycles. Freshly
thawed or recently transported cells are still in a
condition of stress and should not be used
immediately for electroporation.
Low transfection rates Cells are contaminated
with mycoplasma. Mycoplasma prevent successful electroporation
of cells with the Multiporator
® . Cell cultures
must be checked for mycoplasma at regular
intervals (Sec. 4.1.1).
Pulse is too weak. A pulse with a low field strength may be too
weak to permeate the cell membrane. Check
determination of the cell size (Sec. 4.1.4) and
the minimum field strength based on this size
(Table 2). The gap width of the cuvette and the
electroporation temperature must also be
considered.
Problem Possible cause Solution / comments
5 Troubleshooting
5 Troubleshooting
Multipor_Appli_E_poration_en.fm Seite 43 Montag, 30. Januar 2006 2:17 14