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Publication 5423-020-REV 1.0 • www.btxonline.com
Pulse Length
The pulse length is the duration of time the sample is exposed to
the pulse. This is measured as time in ranges from microseconds
to milliseconds. Adjusting this parameter is dependent on the
pulse waveform. The pulse length in a square wave system can be
inputted directly. The pulse length in an exponential decay wave
system is called the “time constant” which is characterized by
the rate at which the pulsed energy (e) or voltage is decayed to
one-third the original set voltage. This time constant is modified
by adjusting the resistance and capacitance (RC) values in an
exponential decay waveform. Time constant calculation T = RC,
where T is time and R is resistance and C is capacitance.
The pulse length works indirectly with the field strength to
increase pore formation and therefore the uptake of target
molecules. Generally, during optimization of parameters an
increase in voltage should be followed by an incremental decrease
in pulse length. When decreasing the voltage, the reverse is true.
Pulse length is a key variable that works hand in hand with voltage
and needs to be considered when optimizing electrical parameters
to maximize the results for a given cell type.
Number of Pulses
Electroporation is typically carried out as a single pulse for most
cell types. However, other cell lines may require multiple pulses
to achieve maximum transfection efficiencies. Usually lower
voltages are used when applying multiple pulses in order to
gradually permeate the cell membranes. This allows the transfer
of molecules while avoiding damage to delicate or whole tissue
samples. This method of multiple pulsing is critical for maximum
gene delivery without causing tissue damage to in vivo, in utero
and explant tissue environments. The use of multiple pulse will
require the optimization of key electrical parameters including
voltage and pulse length. Typically, for in vivo applications the use
of lower voltages between 10 and 100 volts with pulse lengths
ranging 30 to 50 ms provides efficient transfection. The optimal
voltage, pulse length and number of pulses will vary depending on
the cell type and molecule (DNA or RNA) transfected.
Electroporation Buffer
The buffers used for electroporation can vary depending on the
cell type. Many applications use highly conductive buffers such
as PBS (Phosphate Buffered Saline <30 ohms) and HBSS (Hepes
Buffer <30 ohms) or standard culture media which may contain
serum. Other recommended buffers are hypoosmolar buffers in
which cells absorbs water shortly before pulse. This swelling of
the cells results in lowering the optimal permeation voltage while
ensuring the membrane is more easily permeable for many cells
but can be damaging to others. Prokaryotic cells such as bacteria
require the use of high resistance buffers (>3000 ohms). For this
reason proper preparation and washing of the cells is essential
to remove excess salt ions to reduce the chance of arcing. Ionic
strength of an electroporation buffer has a direct affect on the
resistance of the sample which in turn will affect the pulse length
or time constant of the pulse. The volume of liquid in a cuvette
has a significant effect on sample resistance for ionic solutions; the
resistance of the sample is inversely proportional to the volume
of solution and pH. As the volumes are increased resistance
decreases which increases the chance of arcing, Lowering the
volume will increase the resistance and decrease the arc potential.
BTX offers BTXpress High Performance Electroporation Solution,
a low conductance buffer that achieves higher transfection
efficiencies with minimal cell toxicity. The BTXpress buffer is a
single buffer developed to facilitate high efficiency gene delivery
into mammalian cells.
DNA/RNA Concentrations
Electroporation is typically thought of as a nucleic acid (DNA,
mRNA, siRNA and miRNA) transfer method into prokaryotic and
eukaryotic cells. Electroporation is not limited to just nucleic acid
delivery, it can introduce proteins, antibodies, small molecules and
fluorescent dyes.
The standard range of DNA used for transfections is 5 – 20 µg/ml
for most cell types; however in some instances increasing the DNA
concentration as high as 50 µg/ml improves transfection efficiency
without changing other parameters. Determining the optimal
DNA concentration through a DNA titration can be beneficial. The
size of a molecule will have an effect on the electrical parameters
used to transfect the cell. Smaller molecules (siRNA or miRNA)
may need higher voltage with microsecond pulse lengths and
larger molecules (DNA) may need lower voltages with longer pulse
lengths. Buffers such as EDTA or Tris can drastically reduce the
transfection efficiency. Therefore, we recommend resuspending
DNA in distilled water. Finally, electroporating ligation mixtures
into E. coli can cause arcing and reduced transformations. Diluting
the ligation mixture a minimum of 1:5 with diH
2
O, dialysis, or
ethanol precipitation can significantly improve transformation
efficiencies and reduce the potential for arcing.
General Optimization Guide for Electroporation
Protocol Optimization In Vitro
Choose the optimal field strength based on the best conditions observed when
plotting viability versus expression at different field strengths.
