General Optimization Guide

for Electroporation
Electroporation is the application of controlled direct current (DC) electrical pulses which are applied to living cells
and tissues for a short duration of time. The pulse induces a transmembrane potential which causes the reversible
breakdown of the cellular membrane. This action results in the permeation or “pore formation” of the cell membrane
which allows small molecules (such as dye, oligonucleotides or peptides) and large molecules (such as proteins,
DNA and RNA) to be introduced into the cell. During this process the cellular uptake of the molecules continue until
the pores close which can take milliseconds to minutes.
Electrofusion is an expansion of electroporation using different buffers and one or more proprietory alternating current
(AC) pulse(s). Cells are brought together or “aligned” by the use of an AC pulse which causes charges to form on the
cellular membrane (dielectrophoresis) resulting in alignment of cells or pearl-chain (dimer) formation. Following the
AC cellular alignment the DC pulse is applied to induce permeation of the cell membrane. When cells are brought
into contact during electroporation, these cells are induced to fuse. Following this DC pulse the AC pulse is main-
tained to allow complete cell membrane fusion during the recovery period.
Optimization of the electroporation process involves several factors. Choosing the wave form, determining field
strength and adjusting pulse length are just a few critical variables. Other parameters which play a crucial role in
optimization include cell diameter, DNA concentrations, temperature and electroporation buffer.

Wave Forms 1. Cuvette Gap Size

Pulse shape generally falls into two categories, square wave or The distance between electrodes, or “gap size” is important when opti-
exponential decay wave: mizing your electroporation experiment. Field strength is calculated
using voltage divided by gap size. For example, using a 4mm gap
Square wave pulse: Square wave pulses rise quickly to a set
cuvette with 500V would provide a field strength of 1.25kV/cm. If
voltage level, maintains this level during the duration of the set pulse
instead of a 4mm gap cuvette, a 2mm gap cuvette was used, the volt-
length and quickly turns off. This square wave system yields higher
age would have to be reduced by half or 250V in order to maintain the
efficiencies and viabilities in mammalian cells. Square wave EP in
same field strength of 1.25kV/cm. It is possible to derive the voltage
in vivo and ex vivo tissues, embryo’s, cell fusions and plant protoplast
needed to accomplish electroporation if the desired field strength and
applications yield better results in comparison to an exponential decay
gap size are known. The calculation for this is Field strength (kV) multi-
wave system.
plied by gap size (cm) equals voltage. For example, if a user was certain
Exponential decay wave pulse: Exponential decay waves that a 1.25 kV/cm field strength was required in a 1mm gap cuvette the
generate an electrical pulse by allowing a capacitor to completely calculation would be: 1.25kV x 0.1cm= 0.125kV or 125volts.
discharge. A pulse is discharged into a sample the voltage rises rapidly
Example: A field strength of 1.25 kV/cm
to the peak voltage set then declines over time. The powerful exponen-
tial decay wave pulse is routinely used for transformation of gram-nega- 4mm gap cuvette = 500 volts
tive and gram-positive, bacterial, yeast, plant tissues, insect cells and 2mm gap cuvette = 250 volts
some mammalian cells. 1mm gap cuvette = 125 volts

Field Strength 2. Cell Diameter

The field strength is measured as the voltage delivered across an Generally, smaller cell sizes require higher voltages while larger cell
electrode gap and is expressed as kV/cm. Field strength is critical to diameters require lower voltages for successful cell membrane
surpassing the electrical potential of the cell membrane to allow the permeation.
temporary reversible permeation or “pore formation” to occur in the cell
membrane. Three factors should be considered for optimizing field
strength: Cell Cuvette 4mm Cuvette 4mm
Diameter Room Temp. (Volt) 4ºC
1. Cuvette Gap Size
2. Cell Diameter 10 500 Volts 1000 V
3. Temperature 15 350 Volts 700 V
Cell Types Field Strength Ranges 20 250 Volts 500 V

Bacteria/Yeast 3-24 kV/cm 30 180 Volts 360 V

Mammalian 0.25-3 kV/cm 40 130 Volts 250 V

50 100 Volts 200 V
Plant 3-12 kV/cm

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General Optimization Guide
for Electroporation (continued)
3. Temperature Electroporation Buffer
The temperature at which cells are maintained during electroporation The buffers used for electroporation can vary depending on the cell
effects the efficiency of the electroporation for several reasons. For a type. Many applications use highly conductive buffers such as PBS
majority of mammalian cell lines are effectively electroporated at room (Phosphate Buffered Saline <30 ohms) and HBSS (Hepes Buffer
temperature. Samples which are pulsed at high voltage or exposed to <30 ohms) or standard culture media which may contain serum. Other
multiple pulses and long pulse durations can cause the sample to heat recommended buffers are hypoosmolar buffers in which cells absorbs
up. These conditions cause increased cell death and lowers the trans- water shortly before pulse. This swelling of the cells results in lowering
fection efficiency. Maintaining the sample at lower temperatures can the optimal permeation voltage while ensuring the membrane is more
diminish the heating effects on cell viability and efficiency. Since electro- easily permeable for many cells but can be damaging to others.
poration causes the transient formation of pores, keeping the cells at Prokaryotic cells such as bacteria require the use of high resistance
lower temperature following the pulse may allow the pores to remain buffers (>3000 ohms) for this reason proper preparation and washing of
open longer to allow more uptake of the exogenous molecule. Yet lower the cells is essential to remove excess salt ions to reduce the chance of
temperatures on other cell lines can be damaging and cause high cell arcing. Ionic strength of an electroporation buffer has a direct affect on
mortality. This effect is specific to each cell line and should be consid- the resistance of the sample which in turn will affect the pulse length or
ered during optimization studies. The standard pulse voltage used for time constant of the pulse. The volume of liquid in a cuvette has signifi-
cells at room temperature will need to be approximately doubled cant effect on sample resistance for ionic solutions, the resistance of the
for electroporation at 4°C in order to effectively permeate the cell sample is inversely proportional to the volume of solution and pH. As
membrane. the volumes are increased resistance decreases which increases the
chance of arcing, while lowering the volume will increase the resistance
Pulse Length and decrease the arc potential.
The pulse length is the duration of time the sample is exposed to the BTX now offers BTXpress™ High Performance Electroporation Solution,
pulse. This is measured as time in micro to milliseconds ranges. a low conductance buffer that achieves higher transfection efficiencies
Adjusting this parameter is dependent on the pulse generator in use with minimal cell toxicity. The BTXpress buffer is a single buffer devel-
square wave or exponential decay wave. The pulse length in a square oped to facilitate high efficiency gene delivery into mammalian cells.
wave system can be inputted directly. The pulse length in an exponen-
tial decay wave system is called the “time constant” which is character- DNA/RNA Concentrations
ized by the rate at which the pulsed energy (e) or voltage is decayed to
Electroporation is typically thought of as a nucleic acid (DNA, mRNA,
1/3 the original set voltage. This time constant is modified by adjusting
siRNA and miRNA) transfer method into prokaryotic and eukaryotic
the resistance and capacitance (RC) values in an exponential decay.
cells. Electroporation is not limited to just nucleic acid delivery, it can
Time constant calculation T=RC, where T is time and R is resistance
introduce proteins, antibodies, small molecules and fluorescent dyes.
and C is capacitance.
The standard range of DNA used for transfections is 5-20 g/ml for
The pulse length works indirectly with the field strength to increase pore most cell types; however in some instances increasing the DNA con-
formation and therefore the uptake of target molecules. Generally, dur- centration as high as 50 g/ml improves transfection efficiency without
ing optimization of parameters an increase in voltage should be followed changing other parameters. Determining the optimal DNA concentration
by an incremental decrease in pulse length. Decreasing the voltage, the through a DNA titration can be beneficial. The size of a molecule will
reverse is true. Pulse length is a key variable that works hand in hand have an effect on the electrical parameters used to transfect the cell.
along with voltage and needs to be considered when optimizing electri- Smaller molecules (siRNA or miRNA) may need higher voltage with
cal parameters to maximize the results for a given cell type. microsecond pulse lengths and larger molecules (DNA) may need lower
voltages with longer pulse lengths. Buffers such as EDTA or Tris can
Number of Pulses drastically reduce the transfection efficiency. Therefore, we recommend
Electroporation is typically carried out as a single pulse for most cell resuspending DNA in distilled water. Finally, electroporating ligation mix-
types. However, other cell lines may require multiple pulses to achieve tures into E.coli can cause arcing and reduced transformations. Diluting
maximum transfection efficiencies. Usually lower voltages are used the ligation mixture a minimum of 1:5 with diH2O, dialysis, or ethanol
when applying multiple pulses in order to gradually permeate the cell precipitation can significantly improve transformation efficiencies and
membranes. This allows the transfer of molecules while avoiding dam- reduce the potential for arcing.
age 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 ex-plant tissue environments. The use of multiple
pulsing will require the optimization of key electrical parameters including
voltage and pulse length. Typically, for in vivo applications the use of
lower voltages between 10-100 volts with pulse lengths ranging
30-50msec 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.

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