Lonza 4D-Nucleofector - Troubleshooting Guide; Addressing Suboptimal Transfection Results

36 | 4D-Nucleofector
®
Manual
3 Troubleshooting
3.1 Suboptimal transfection results
The following troubleshooting guidelines may be helpful
if experiments using the 4D-Nucleofector
®
System do not
provide the expected results. The comments are intended
to help optimize experimental conditions. If you require
futher help, please contact our Scientific Suppot Team.
Issue Possible error Solution
Low survival rate Cells were kept in Nucleofector
®
Solution
too long
Transfer cells immediately into pre-warmed medium as recommended in the
optimized protocol.
Cells were damaged by harvesting procedure or
through handling
Avoid harsh conditions during cell harvesting, especially centrifugation at
higher speed or overexposure to trypsin.
Pipette cells smoothly as they are quite stressed already. Use a plastic pipette
as recommended in the optimized protocols.
Cells culture conditions were suboptimal Cells should be viable and in culture for several passages. Avoid excessive cell
densities or cell confluencies since this may decrease cell viability post
Nucleofection
®
Procedure. For futher details, please refer to the dedicated
optimized protocol.
Nucleocuvettes
®
Vessels were reused It is strongly recommended to use the Nucleofection
®
Vessels only once, be-
cause the high voltage pulses that are applied drastically affect their
physical integrity.
Poor DNA quality DNA used for Nucleofection
®
Experiments should be of high purity. We
strongly recommend endotoxin-free preparation of the DNA. Do not use
procedures involving phenol /chloroform treatment.
Low efficiency DNA amount is too low Depending on cell type and Nucleofection
®
Vessel a cetain DNA amount per
sample is recommended (for details, please refer to the respective optimized
protocol). If both, transfer efficiency and cell motality are low, the DNA
amount could be increased. Increasing the amount of DNA may result in in-
creased transfection efficiency but also in increased cell motality.
Cell number in Nucleofection
®
Sample too high
or too low
Please use the cell numbers recommended in the dedicated
optimized protocol.
Poor DNA quality DNA used for Nucleofection
®
Experiments should be of high purity. We
strongly recommend endotoxin-free preparation of the DNA. Do not use
procedures involving phenol /chloroform treatment.
System unable to stat Buffer battery too low to stat the system If the device is not used for longer periods, it will require occasional recharging
to avoid battery discharge and ensure functionality. To avoid a deep discharge
of the battery and loss of data, switch on the system at least once a month.
If the problem cannot be resolved, please call Lonza’s
Scientific Suppot Team
Europe
Phone: + 49 221 99199 400
E-mail: scientific.suppot.eu@lonza.com
Noth America
Phone: 800 521 0390 (toll free)
E-mail: scientific.suppot@lonza.com
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