Electroporation has transformed the way in which scientists work, allowing the delivery of genetic material, proteins, drugs or other molecules into mammalian, insect, fungal, plant and bacterial cells. Nevertheless, as with all laboratory techniques, it is not without its fair share of pitfalls and disappointments.
Therefore, it is important to adopt best practices and optimize your experimental conditions to fit your cell type and application.
Download this guide to discover tips on:
Preparing your samples
Selecting your parameters
Perfecting your electroporation protocol
How to Guide
How To Optimize Your Electroporation
Carl Robinson, PhD
The use of electroporation as a tool to deliver materials into cells was first described in 1972 by
Neuman and Rosenheck. The process, which uses short electrical impulses to generate reversible
holes in the cell membrane, has subsequently been used to deliver genetic material (DNA and
RNA), proteins, drugs or other molecules into mammalian, insect, fungal, plant and bacterial cells.
Electroporation has transformed the way in which scientists work. Nevertheless, as with all laboratory
techniques, it is not without its fair share of pitfalls and disappointments. Therefore, it is of utmost
importance to follow good practices and optimize your experimental conditions to fit your cell type and
This guide will provide some tips and advice on how to avoid common problems with your
electroporation experiments and optimize your protocols and parameters to get the best results.
Preparing your samples
This one’s just right
First, make sure the cells you wish to use are suitable for electroporation, otherwise you will be onto
a loser from the start. They need to be healthy, free from mycoplasma or other unwanted infections,
free from contaminants and in an appropriate growth phase, typically log phase. When it comes to
the electroporation itself, for experimental success, it’s important that cells are kept at the right
temperature for the cell type being used – not too hot, not too cold, just right. For many bacterial cells,
this means keeping them chilled to slow down their biological processes, reducing harm to the cells
during the experiment. However, other cell types, including many mammalian cell lines, do not tolerate
chilling well and therefore should be kept warm. It is important to do your homework and find out what
is most appropriate for your cells.
Finding the sweet spot
Check the literature to see if others have made recommendations about the number or ratio of
your cells of interest to use if they have performed similar experiments. This can help to give you
a starting point from which to work out the number of cells and amount of the target molecule you
wish to deliver to achieve optimal efficiency. Initially, this is likely to require a bit of trial and error but
remember to do this in a structured way. Use a dilution series of the target with different set amounts
of cells to find the sweet spot. Too few cells and the yield of successfully transformed/transfected cells
will inevitably be low and cell types that rely on cell-to-cell contact may exhibit poor growth after the
experiment. Too high a density of cells and uptake of the target molecule may be inhibited. There isn’t a
“right” concentration; you will need to optimize for your particular situation.
HOW TO OPTIMIZE YOUR ELECTROPORATION 2
How to Guide
Scale to suit
Electroporation cuvettes are where the magic happens and they come in a range of sizes to suit
differing experimental needs. Consider the amount of material you have available for your experiment
– both of cells and insert – and the amount you require at the end of the process and choose
accordingly. When it comes to setting up your protocol on the electroporation machine, remember to
indicate the correct cuvette size – as the gap distance between the conductive plates will differ – so
that it is able to deliver an appropriate impulse for your reaction volume.
Salts are an electroporator’s worst friend
Salts can be very problematic in electroporation reactions, causing harmful arcing in the machine
and consequently cell death. It is highly recommended to wash and resuspend your cells with an
appropriate medium to remove excess salts before proceeding. Sucrose is a popular choice for
bacterial cells, although specialist electroporation buffers are also a great option and are often used
for mammalian cells. This will also help to remove contaminants such as undesirable proteins that can
reduce efficiency. You may also need to purify your insert solution too for the same reason, depending
on what you have resuspended it in. The ionic strength of your sample buffer will impact its electrical
properties so bear this in mind when setting parameters.
Selecting your parameters
Turning your attention to the electroporation equipment, there are a number of factors to consider. This
includes the waveform used, pulse pattern, pulse duration, voltage and capacitance. Many instruments
come with several preset protocols, but it’s important to understand what changing each parameter
will do. This will help you optimize experiments that are not performing as well as they ought to and
will come in useful if there are no preset protocols that suit your need. Conditions may vary from
instrument to instrument too, so optimal conditions for one may not hold true for another.
Two waveforms are generally used for electroporation – square waves and exponential decay waves.
Square waves rapidly rise to their target voltage, maintain it for their specified duration and then
rapidly reduce to nothing again and are popular for mammalian cell electroporation. With exponential
decay waves on the other hand, the voltage rises rapidly to the target voltage but then declines
gradually over time as a capacitor discharges. This form is more commonly used for bacterial, yeast,
plant and insect cells and some mammalian cells.
For some cells, a single pulse is enough to generate usable pores in the cell membrane. However,
for others, a series of multiple, lower voltage pulses produce pores more reliably and effectively. The
voltage is normally reduced where multiple pulses are used as otherwise permanent damage may be
caused to the cells, reducing cell viability. Multiple, lower voltage pulses may also be gentler on fragile
cells compared to a single large shock.
Pulse length, resistance and capacitance
The electrical pulses used in electroporation typically last micro- to milliseconds. For square waves,
this is a simple “on and off”, which can be set directly. However, for exponential decay waves, it is a little
more complex and is referred to in relation to the time constant (T).
HOW TO OPTIMIZE YOUR ELECTROPORATION 3
How to Guide
Altering the resistance and capacitance values will determine how long the pulse lasts, as these
parameters impact the rate of discharge. When voltage is increased, pulse duration is typically
decreased and vice versa to prevent irreparable damage to the cells.
As a rule of thumb, pulse times need to be increased where chilled cells are used over room
temperature cells to enable pores to be opened effectively in the membrane.
The voltage delivered across the gap between the conductive electrical plates of the electroporation
cuvette is known as the field strength and is measured in kV/cm. To get the same pulse, the
voltage must be reduced in line with smaller cuvette gap sizes. Cell diameter is also an important
consideration when choosing a pulse voltage. Generally speaking, the smaller the cell, the higher the
voltage required to successfully create pores.
Consider your parameters, particularly if you are performing the electroporation on room temperature
cells rather than chilled cells. High voltages and long or multiple pulses can cause the sample to heat
up and may reduce cell viability.
Ready to go!
Dry the cuvette
If you are ready to give your selected conditions a test run, there are still more factors to consider.
This may sound basic, but it is vitally important that you ensure the cuvette is dry on the outside. This
is particularly pertinent when a cuvette has been kept chilling on ice; a thorough wipe down with
some lab tissue will suffice. Failure to do so can result in arcing when the electrical pulse is delivered,
potentially damaging the equipment and definitely frying your sample!
Treat them well
A successful electroporation experiment doesn’t end at the “zap”. It’s important to treat your cells well
after the event. They’ve just had a shock, quite literally, so will be in a fragile and vulnerable state.
As with all living things, they require nurturing back to health, which means providing them with the
optimal conditions for growth, including factors such as temperature and nutrition. For some cell
types – including many bacterial cells – keeping everything cold was important during the preparation
and electroporation steps, but now is the time to warm them up again. Try not to do this too suddenly
though, as abrupt changes can result in higher rates of cell death and therefore poorer experimental
results. For example, adding cold media to the cold reaction mix and then warming them up gradually
in the incubator may be preferable to the sudden shock of bathing your cold cells in warm media.
Again, this varies between cell types so find out what is best for your cells. Delays in attending to your
cells after electroporation can rapidly reduce viability so make sure you don’t leave them hanging
around. The precise conditions to enable the cells to start dividing once more will vary depending upon
your target cell type but are likely to be similar to those you use normally to obtain optimal growth.
Where selective agents are used, it is generally advisable to refrain from including these immediately
following electroporation to allow the cells some time to recover first. For bacteria, this may mean
waiting a matter of hours whereas for mammalian cells a day or two’s pause is advisable.
HOW TO OPTIMIZE YOUR ELECTROPORATION 4
How to Guide
Efficiency is key
If you are in the process of optimizing an electroporation protocol, compare the outcomes between
the conditions tested. Look at overall cell survival and what percentage of those surviving cells have
been transformed/transfected successfully. You should expect to kill a fairly large percentage of your
cells. After all, the electrical pulse needs to be strong enough to disrupt the cell membrane, otherwise
the pores won’t form, but you need to get enough cells out to make the experiment viable. If the
overall cell numbers are low, are you putting enough cells in at the start or are they dying during the
electroporation process? If you are getting plenty of viable cells but they do not contain the molecule
you are trying to insert then are you adding enough insert to the reaction or are the electroporation
conditions suboptimal, leading to poor pore formation or insert entry? Try adjusting parameters
and repeating the process to work through the troubleshooting steps, one by one if possible, so you
can discern which parameter is responsible for giving the changes you are seeing. It is possible to
undertake the electroporation process with a reporter, such as a luciferase, to help with parameter
optimization; but this can be less convenient than merely checking cell survival if your lab is not set up
One final thing to remember, optimization can be a time-consuming task so only do it if you really
need to. If you are performing a one-off experiment and one correct transformant/transfectant is all
you need, for example, then consider if it is worth the time investment. Sometimes, just enough is
With all the points discussed above in mind, this guide should give you a head start along the road to
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