Bio-Rad MicroPulser™ Electroporator - Manual (Page 8)

The purpose of the 30 ohm series resistor in the MicroPulser is to protect the instrument
circuitry should arcing occur. Under normal operation, when samples are in high resistance
media, this resistor will not affect the voltage applied to the sample. However, this resistor will
significantly decrease the voltage applied to the sample if the resistance of the sample is low.
The fractional drop in voltage applied to the sample is given by
R
30
/ (R
30
+ R
sample
)
When R
sample
is 600 ohms, there is a 5% voltage drop to the sample [30 / (30 + 600) =
0.048]. For this reason, electroporation with the MicroPulser should not be performed in
solutions with a resistance of less than ~600 ohms. This includes samples in which the growth
medium was not adequately removed from the cells, DNA samples containing salt contributed
by residual sodium chloride, or ligation mixtures. The MicroPulser is able to measure the
resistance of the sample and will not pulse into very low resistance media.
2.2 Manipulation of Instrument Parameters
Several parameters on the MicroPulser may be altered to achieve maximum
transformation efficiency. These include the field strength, E, the time constant, τ, and the
width of a truncated exponential decay pulse. The field strength may be manipulated in two
ways. First, voltages between 200 and 3000 V may be set directly on the MicroPulser. This
parameter is the most easily controlled. The process of varying the voltage while keeping all
other conditions unchanged is the basis for most electroporation optimization procedures.
Second, using cuvettes with different electrode gap widths permits a means of changing the
field strength. For electroporation of microorganisms, 0.1 and 0.2 cm gap cuvettes are most
often used. Electroporation of E. coli is generally carried out at a voltage of 1.8 kV
(E = 18 kV/cm) when electroporating cells in 0.1 cm cuvettes and at a voltage of 2.5 kV
(E = 12.5 kV/cm) when electroporating cells in 0.2 cm cuvettes. These electroporation
conditions are pre-programmed into the MicroPulser as programs Ec1 (V = 1.8 kV) and Ec2
(V = 2.5 kV) in the bacterial settings menu. In addition, a third program, Ec3 in the bacterial
settings menu, delivers a voltage of 3.0 kV (E = 15 kV/cm in 0.2 cm cuvettes) which we have
found results in even higher transformation efficiency compared to electroporation at 2.5 kV.
The time constant may be altered by changing the sample resistance. The sample
resistance may be manipulated in two ways. First, increasing the salt or buffer concentration
of the electroporation media decreases the resistance of the sample, and vice versa, resulting
in a change in the time constant. Second, the volume of the sample in the cuvette is inversely
proportional to the resistance of the sample; decreasing the sample volume increases the
sample resistance. This effect of volume on sample resistance is most noticeable in low
resistance media. These effects are discussed further in Section 3.3.
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