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Ingenio® EZporator® Electroporation System
Product User Manual
| Instructions for MIR 51000
Experimental Troubleshooting: High Cellular Toxicity
Mirus Bio
LLC
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(844.647.8724)│
Direct: +1.608.441.2852
Problem
Solution
Voltage setpoint too
high for cell type
Decrease the voltage by increments of 10 V until viability improves.
Cells not transferred
immediately to cul-
ture vessel contain-
ing complete growth
medium
Transfer the cells from each cuvette to a culture dish containing warm
complete culture medium immediately after each electroporation.
Endotoxin
-
contaminated plas-
mid DNA
Use highly purified, sterile, endotoxin and contaminant
-
free DNA for
electroporation.
We recommend using Mirus Bio MiraCLEAN® Endotoxin Removal Kit (MIR
5900) for removal of any traces of endotoxin from your DNA preparation.
Alternatively, use cesium chloride gradient or anion exchange purified
DNA which contains levels of endotoxin that do not harm most cells.
DO NOT use DNA prepared using miniprep kits as it might contain high
levels of endotoxin.
DNA preparation has
too much salt
If DNA was prepared using an ion exchange column with a final ethanol
precipitation step, we recommend exchanging the DNA solution to a salt
-
free or low salt solution, e.g. elute the DNA in water and add 5 mM NaCl.
Excessive amounts of
DNA in the final
electroporation mix
Reduce the amount of DNA used for electroporation. DNA concentrations
as low as 5 µg/ml of the final electroporation volume can be used.
Compare toxicity levels against a cells + Ingenio® Electroporation Solution
control to assess the effects of the DNA transfected. If you still see toxici-
ty, it is likely due to the cell mixture being too concentrated or presence
of too many lysed cells.
Expressed target
gene is toxic to cells
Compare toxicity levels against a cells
-
alone control and cells electro-
porated with an empty vector to assess the cytotoxic effects of the target
protein being expressed.
If a lower level of target gene expression is desired in your electro-
poration experiments, consider reducing the amount of target plasmid. If
necessary, maintain the optimal DNA concentration (20 µg) by using
carrier DNA such as an empty cloning vector.
Knockdown of an
essential gene
If the electroporated siRNA is directed against a gene that is essential to
the cell, cytotoxicity may be observed due to knockdown of the target
gene. Include a control with non
-
targeting siRNA to compare the cytotox-
ic effects of the gene being knocked down.
Cell morphology has
changed
Mycoplasma contamination can alter cell morphology and affect electro-
poration efficiency. Check your cells for Mycoplasma contamination. Use
a fresh frozen stock of cells or use appropriate antibiotics to eliminate
Mycoplasma.
A high or low cell passage number can make cells more sensitive and
refractory to electroporation. Maintain a similar passage number be-
tween experiments to ensure reproducibility.
