Bio-Rad Gene Pulser® Electroporation Buffer - Manual (Page 2)

Optimization
Determining the optimum electroporation conditions is essential to maximize the transfection efficiency of siRNAs, to obtain the
best gene silencing results, and to minimize cellular toxicity. The same is true for the delivery of plasmid DNA.
Optimize parameters for every cell line using the guidelines in Table 1.
* See Table 2 for maximum values.
** If using a 0.2 cm cuvette, reduce the voltage by 50%.
Transfection Protocol
1. Harvest and count the cells.
a. The cells should be passaged the day before electroporation. All cell types should be harvested when they are
actively growing. If working with adherent cells, trypsinize the cells to detach them, add growth medium, and
then pellet the cells. If working with suspension cells, pellet the cells.
b. After pelleting the cells, remove the medium and wash the cells once with PBS, by carefully pipetting them.
Take an aliquot and count the cells.
2. Prepare the cells for electroporation.
a. Aliquot the number of cells needed to perform the experiment. For adherent cells, we recommend using 1 x 10
6
cells/ml, but we have successfully used 0.5–5 x 10 6 cells/ml. For suspension cells, we recommend using 2–3 x 10 6
cells/ml.
b. Pellet the cells.
c. Aspirate the PBS and resuspend the cells in the appropriate volume of electroporation buffer reagent (1 ml per
1 x 10 6 of adherent cells, and 1 ml per 2–3 x 10 6 of suspension cells).
d. Add nucleic acid. For siRNA electroporation, use 10–100 nM of siRNA. For plasmid DNA electroporation, use
5–20 µg/ml.
3. Electroporate cells.
a. Choose the proper vessel for your sample size (see Table 2).
b. Electroporate the sample using optimized conditions.
c. Transfer cells to tissue culture dishes containing growth media.
d. Incubate cells at 37°C in a humidified CO
2
incubator until ready to be assayed. After 24 hr, change the growth
medium.
4. Assess transfection efficiency.
a. Fluorescently labeled siRNAs can be used to determine the transfection efficiency for siRNA delivery.
Transfection efficiency can be measured by fluorescent microscopy or flow cytometry.
b. For plasmid delivery, the transfection efficiency can be determined by electroporating plasmids expressing
reporter genes such as GFP or b-galactosidase.
Table 2. Proper vessel based on sample size.
Sample Volume Vessel
100–200 µl 0.2 cm cuvette
400–800 µl 0.4 cm cuvette
100–200 µl 96-well plate
500–800 µl 24-well plate
1.0–1.5 ml 12-well plate
Table 1. Electroporation optimization guidelines.
Parameter Optimization range
Voltage Square waveforms 50–400 V (in 10 V increments)
Exponential waveforms 100–350 V (in 50 V increments)
Pulse length
(for square waveforms) 10–25 ms
Capacitance
(for exponential waveforms) 500–1000 µF
Vessel size* 0.4 cm 2 cuvette** or
96-, 24-, or 12-well electroporation plate
Cell density Adherent cells 1–5 x 10
6
cells/ml
Suspension cells 2–10 x10 6 cells/ml
siRNA concentration 10–100 nM