Troubleshooting,
Continued
Problem
Cause
Solution
Air bubbles in the
Neon
™
Tip
Avoid any air bubbles in the Neon
™
Tip while aspirating the
sample.
High voltage or pulse
length settings
Reduce the voltage or pulse length settings.
Arcing (sparks)
Accidentally used
salt-precipitated
DNA
Do not precipitate DNA with ethanol to concentrate DNA as
it can cause arcing due to salt contamination.
Poor DNA quality
Use high quality plasmid DNA for transfection (see page 13
for guidelines and recommendations on DNA quality).
Cells are stressed or
damaged
Avoid severe conditions during cell harvesting especially
high speed centrifugation and pipette cells gently.
Avoid using over confluent cells or cells at high densities as
this may affect the cell survival after electroporation.
After electroporation, immediately plate the cells into
prewarmed culture medium
without antibiotics
.
Low cell
survival rate
Multiple use of the
same Neon
™
Tip
Do not
use the same Neon
™
Tip for electroporation for more
than 2 times because the repeated application of electric
pulses reduce the tip quality and impairs their physical
integrity.
Poor optimization of
electrical parameters
Perform optimization for your cell type as described on
page 22.
Poor plasmid DNA
quality or the
plasmid DNA is low
Use high quality plasmid DNA for transfection (see page 13
for guidelines and recommendations on DNA quality).
Start with 0.5 μg plasmid DNA per sample.
Incorrect cell density
Cell densities >3 × 10
5
or <5 × 10
4
per sample drastically
reduces transfection efficiency. Use 5 × 10
4
–
1.5 × 10
5
cells per
10 μL per sample.
Low transfection
efficiency
Mycoplasma
contaminated cells
Test cells for
Mycoplasma
contamination.
Start a new culture from a fresh stock.
Inconsistent cell
confluency or
passage number
Always use cells with low passage number and harvest cells
with comparable confluency levels.
Non-
reproducible
transfection
efficiency
Multiple use of
Neon
™
Tip and
Neon
™
Tube
Do not
use the same Neon
™
Tip for more than
2 times
because the repeated application of electric pulses reduce the
tip quality and impairs their physical integrity.
Do not use the same Neon Tube for more than 10 times.
Always use a new Neon
™
Tip and Neon
™
Tube for different
plasmid DNA samples to avoid any cross-contamination.
High energy
error
Used high electrical
parameters
Set lower voltage or duration.
32
