Mirus Ingenio EZporator MTR 51000, Руководство пользователя

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Ingenio® EZporator® Electroporation System
Product User Manual | Instructions for MIR 51000
 Experimental Troubleshooting: Low Electroporation Efficiency
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Problem Solution
Cell density not
optimal at time of
electroporation
Determine the best cell density for each cell type to maximize electro-
poration efficiency. For most suspension cells, a cell density of 1 × 10
7
cells/ml is recommended for electroporation. For adherent cells, a range
of 1
- 5 × 10
6
cells/ml is recommended. Use of higher or lower densities
may increase cell viability depending on cell type.
Cells not actively
dividing at the time
of electroporation
Passage cells at least 18 – 24 hours before electroporation to ensure that
the cells are actively dividing and reach optimal cell density at the time of
electroporation.
Suboptimal DNA
concentration
Confirm DNA concentration and purity. Use plasmid DNA preps that have
an A
260/280
absorbance ratio of 1.8 - 2.0.
The optimal DNA concentration generally ranges between 5 - 50 µg/ml of
final electroporation volume. Start with 20 µg/ml of total electroporation
volume.
Low quality plasmid
DNA
Use highly purified, sterile, endotoxin and contaminant - free DNA for
transfection. Do not use DNA prepared using miniprep kits as it might
contain high levels of endotoxin.
Incorrect vector
sequence
If you do not observe expression of your target insert, verify the se-
quence of the plasmid DNA.
Proper experimental
controls were not
included for plasmid
delivery
To verify efficient electroporation, deliver a positive control such as a
luciferase or green fluorescent protein (GFP) encoding plasmid.
To assess delivery efficiency of plasmid DNA, use Mirus Bio Label IT
®
Tracker ™ Intracellular Nucleic Acid Localization Kit to label the target
plasmid or use Mirus Bio prelabeled Label IT® Plasmid Delivery Controls.
Suboptimal siRNA
concentration
The optimal siRNA concentration generally ranges between 250 - 750 nM
final concentration. Use 250 nM siRNA as a starting point.
Incorrect siRNA
sequence
Ensure that the sequence of the siRNA is correct for the gene of interest.
More than one sequence may need to be tested for optimal knockdown
efficiency and to ensure proper targeting.
Poor quality of siRNA
Avoid siRNA degradation by using RNase - free handling procedures and
plastic ware. Degradation of siRNA can be detected on acrylamide gels.
Proper controls were
not included for
siRNA delivery
Recommended controls include: (1) Cells alone, (2) Serum - free
Ingenio
®
Electroporation So a non - targeting siRNA.
To verify efficient transfection and knockdown, deliver a siRNA targeted
against a ubiquitous gene, e.g. GAPDH or Lamin A/C, followed by target
western blotting or mRNA quantification.
Electroporation
incubation time
Determine the optimal electroporation incubation time for each cell type
and experiment. Test a range of incubation times (e.g. 12
- 72 hours). The
best incubation time is generally 24 - 48 hours.