Gemini Series Electroporator User’s Manual
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8
Pulse Length
The pulse length is the duration of time the sample is exposed
to the pulse. This is measured as time in micro to milliseconds
ranges. Adjusting this parameter is dependent on the pulse wave
form. 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 1/3 the
original set voltage. This time constant is modified by adjusting
the resistance and capacitance (RC) values in an exponential decay
wave form. 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 10-100 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.
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 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, while lowering the volume
will increase the resistance and decrease the arc potential.
BTX now 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 5-20g/ml for most cell types; however in some
instances increasing the DNA concentration as high as 50g/
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 diH2O, dialysis, or ethanol precipitation can
significantly improve transformation efficiencies and reduce the
potential for arcing.
General Optimization Guide for Electroporation