Eppendorf Multiporator - Electroporation - Manual (Page 20)

44
Pulse is too short. Extend the pulse length to increase permeability
of the cell membrane, which can lead to a
higher transfection rate. Note that the optimal
pulse length is affected by the temperature at
which electroporation is carried out.
DNA concentration
is too low / too high. If the transfection rate is too low and the viability
of the cells is high, the plasmid concentration
may be increased.
Note, however, that an increased plasmid
concentration may lead to a higher transfection
rate only within a limited concentration range of
the plasmid.
Large plasmids are used. The size of the plasmid can greatly affect the
transfection rate. When large plasmids are
used, it may be necessary to increase the
permeation of the membrane by applying
pulses with a higher field strength. Be careful as
this may also lead to a higher cell mortality rate.
Cell density is too high. If cell densities are too high during
electroporation, the homogeneity of the
electrical field can no longer be guaranteed.
Reduce the cell concentration in the cuvette to
1 – 3 x 10
6 cells/ml or lower.
Electroporation preparation
contains EDTA or endotoxins. EDTA and endotoxins have a cytotoxic effect.
They are often introduced into the electropora-
tion preparation when DNA is added (e.g. in
TE buffer). Remove EDTA by carrying out a
buffer exchange on bidistilled H
2
0. Endotoxins
can be removed using "endotoxin-free" plasmid
preparation kits.
Incubation period for gene
expression is too short. Following transfection, different cell types
require different incubation periods in order to
reach their maximum expression rate.
Expression should be checked again at a later
point in time.
Problems with
the reporter assay. Include positive controls which indicate that the
reporter system is working properly.
Non-reproducible results Washing procedure prior
to electroporation was
not thorough enough. Traces of medium can have a severe effect on
the conductivity of the electroporation buffer,
and thus on the transfection result. Therefore,
the medium in the supernatant must be
thoroughly removed during the washing
procedure.
Problem Possible cause Solution / comments
5 Troubleshooting
5 Troubleshooting
Multipor_Appli_E_poration_en.fm Seite 44 Montag, 30. Januar 2006 2:17 14