BTX ECM630 - Buffer Selection and DNA Concentration

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Publicaon 5423-020-REV 1.0 • www.btxonline.com
Pulse Length
The pulse length is the duraon of me the sample is exposed to
the pulse. This is measured as me in ranges from microseconds
to milliseconds. Adjusng this parameter is dependent on the
pulse waveform. The pulse length in a square wave system can be
inpued directly. The pulse length in an exponenal decay wave
system is called the “me 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 me constant is modied
by adjusng the resistance and capacitance (RC) values in an
exponenal decay waveform. Time constant calculaon T = RC,
where T is me and R is resistance and C is capacitance.
The pulse length works indirectly with the eld strength to
increase pore formaon and therefore the uptake of target
molecules. Generally, during opmizaon 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 opmizing electrical parameters
to maximize the results for a given cell type.
Number of Pulses
Electroporaon is typically carried out as a single pulse for most
cell types. However, other cell lines may require mulple pulses
to achieve maximum transfecon eciencies. Usually lower
voltages are used when applying mulple pulses in order to
gradually permeate the cell membranes. This allows the transfer
of molecules while avoiding damage to delicate or whole ssue
samples. This method of mulple pulsing is crical for maximum
gene delivery without causing ssue damage to in vivo, in utero
and explant ssue environments. The use of mulple pulse will
require the opmizaon of key electrical parameters including
voltage and pulse length. Typically, for in vivo applicaons the use
of lower voltages between 10 and 100 volts with pulse lengths
ranging 30 to 50 ms provides ecient transfecon. The opmal
voltage, pulse length and number of pulses will vary depending on
the cell type and molecule (DNA or RNA) transfected.
Electroporaon Buer
The buers used for electroporaon can vary depending on the
cell type. Many applicaons use highly conducve buers such
as PBS (Phosphate Buered Saline <30 ohms) and HBSS (Hepes
Buer <30 ohms) or standard culture media which may contain
serum. Other recommended buers are hypoosmolar buers in
which cells absorbs water shortly before pulse. This swelling of
the cells results in lowering the opmal permeaon voltage while
ensuring the membrane is more easily permeable for many cells
but can be damaging to others. Prokaryoc cells such as bacteria
require the use of high resistance buers (>3000 ohms). For this
reason proper preparaon and washing of the cells is essenal
to remove excess salt ions to reduce the chance of arcing. Ionic
strength of an electroporaon buer has a direct aect on the
resistance of the sample which in turn will aect the pulse length
or me constant of the pulse. The volume of liquid in a cuvee
has a signicant eect on sample resistance for ionic soluons; the
resistance of the sample is inversely proporonal to the volume
of soluon 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 potenal.
BTX oers BTXpress High Performance Electroporaon Soluon,
a low conductance buer that achieves higher transfecon
eciencies with minimal cell toxicity. The BTXpress buer is a
single buer developed to facilitate high eciency gene delivery
into mammalian cells.
DNA/RNA Concentraons
Electroporaon is typically thought of as a nucleic acid (DNA,
mRNA, siRNA and miRNA) transfer method into prokaryoc and
eukaryoc cells. Electroporaon is not limited to just nucleic acid
delivery, it can introduce proteins, anbodies, small molecules and
uorescent dyes.
The standard range of DNA used for transfecons is 5 – 20 µg/ml
for most cell types; however in some instances increasing the DNA
concentraon as high as 50 µg/ml improves transfecon eciency
without changing other parameters. Determining the opmal
DNA concentraon through a DNA traon can be benecial. The
size of a molecule will have an eect 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. Buers such as EDTA or Tris can drascally reduce the
transfecon eciency. Therefore, we recommend resuspending
DNA in dislled water. Finally, electroporang ligaon mixtures
into E. coli can cause arcing and reduced transformaons. Dilung
the ligaon mixture a minimum of 1:5 with diH
2
O, dialysis, or
ethanol precipitaon can signicantly improve transformaon
eciencies and reduce the potenal for arcing.
General Optimization Guide for Electroporation
Protocol Opmizaon In Vitro
Choose the optimal eld strength based on the best conditions observed when
plotting viability versus expression at different eld strengths.