Bio-Rad Gene Pulser MXcell™ Electroporation System - Manual (Page 55)

Gene Pulser MXcell™ System Manual | Factors Affecting Electroporation
46
By changing the capacitor of the instrument or by changing the resistance of the circuit, the
time constant may be readily changed. For resistors connected in parallel, the total
resistance of the circuit is given by the following equation:
R Τ = (R
sample
* R
PC
) / (R
sample
+ R
PC
)
When the sample resistance is much greater than the parallel resistor in the PC module
(R
sample
>> R
PC
), the latter is the primary determinant of the resistance of the circuit, and
R Τ
≅ R
PC
. Therefore, the parallel resistance reduces the resistance of the circuit thereby
reducing the time constant of the circuit.
When low-resistance media are used (e.g., high ionic-strength media such as PBS or
growth media used for most mammalian cells), the time constant is most easily manipulated
by selecting the proper capacitor in the Gene Pulser MXcell system. Additionally, changing
the volume of low-resistance media in the cuvette will alter the resistance of the circuit
(resistance is inversely proportional to volume).
Square Wave Pulses
Truncating the pulse from a capacitor after discharging it into the sample generates square
wave pulses. The ideal square wave pulse has the same voltage at the end as at the
beginning of the pulse (Figure 11 on page 47). However, when using a charged capacitor to
produce this waveform (as is done in all commercially available electroporation
instruments), the voltage at the end of the pulse, V
t
, is always less than the voltage at the
beginning of the pulse, V
o
. This is because when the switch is closed across a charged
capacitor, maximum current instantaneously flows through the circuit and gradually falls to
zero. To produce a square wave, the pulse is terminated at some time t, following discharge
of the capacitor. This time (t) is termed the pulse length. The longer the pulse length, the
greater is the difference in voltage between the beginning and the end of the pulse. This
voltage decay may be determined from the following equation:
ln (V
o
/ V
t
) = t / (R C)
The decrease in voltage that occurs with a square wave pulse is inversely related to both
the capacitance of the instrument and the resistance of the sample. The decrease in voltage
at the end of the pulse is termed droop. The fractional decrease in voltage is determined by
the following equation:
Fractional Voltage Decrease (% droop) = (V
o
- V
t
) / V
o
Combining two equations the previous two equations results in the following equation:
ln [1 / (1 - % droop)] = t / (R C)
In order for the pulse to most closely approximate a true square wave, droop must be
minimized (i.e., V
t
= V
o
and V
o
- V
t
= 0). Experimentally, this is achieved by choosing the
highest values for R and C. For any given sample, R may be considered a constant. For
each selected protocol, C may also be considered a constant. Therefore, for the same
sample, as pulse length increases, droop also increases. However, increasing sample
resistance reduces the droop at any given pulse length. Increasing the sample resistance
may be accomplished by the following conditions:
• Reducing the temperature of the sample
• Reducing the ionic concentration of the solution
• Reducing the volume of liquid in the electroporation cuvette in the case of low-
resistance media.