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Rectangular Pulses
Voltages of 2 V to -4 V are sufficient for low molecular weight substances such
as Lucifer yellow, Alexa dyes, siRNA's, etc. The pulse polarity will have to be
adjusted to be the same as that of the molecules to be delivered. The two
protocols outlined below have been shown to work.
Protocol 1:
Train of 100 Hz for 3 seconds
Pulse amplitude: 2 V to -4 V
Pulse duration: 1 ms
Protocol 2:
Single pulse
Pulse amplitude: -3 V to -4 V
Pulse duration: 300 ms
For small to medium size genes (5 kb to 14 kb size range), a rectangular pulse
should be tried initially. Voltages of -5 V to -10 V are useful for inserting large
DNA molecules. For genes ranging from 4 to 5 kb -5.5 V to -6 V is sufficient.
Larger voltages are required for larger genes. To date, 14 kb is the largest gene
that has been successfully electroporated. In general, expression of the larger
genes is problematic. As always, the polarity of the voltage pulse must be the
same as the net charge on the gene.
Start with a pulse voltage of -6 V and width of 1 ms. Set the pulse frequency to
100 Hz and the train duration to three seconds. Use a duty cycle of 10% and a
total on-time of 300 ms. If these parameters are ineffective, but the cells still
seem healthy after the train is complete, consider increasing the voltage
stepwise to -10 V. Cell viability may deteriorate as the voltage approaches
-10 V. Instead of increasing the pulse amplitude try changing two other
parameters:
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