Optimization Protocol for DNA and siRNA,
Continued
18-well
Optimization
Protocol for
Primary
Suspension Blood
Cells—Day One
1.
Make sure you have cells prepared as described on pages 16–17, have the
DNA or siRNA, and prepare 18-wells of a 24-well plate containing 0.5 mL
culture medium with serum and
without antibiotics
to transfer the
electroporated cells. Prepare enough cells and plasmid DNA or siRNA for
at least 20 transfections.
2.
For each electroporation sample using the
10 μL Neon
™
Tip
in
18-wells of a
24-well plate
, see table below.
Cell Type
Cell no.
DNA
siRNA
Resuspension
Buffer T
Primary Blood
Suspension Cells
2 × 10
5
/well
1 μg DNA/well
20 μg/plate
100 pmol in 10 μL tip
200 nM per well
10 μL/well
180 μL/plate
3.
Set up a Neon
™
Tube with 3 mL Electrolytic Buffer E into the Neon
™
Pipette Station and Neon
™
Tip containing the cell-DNA/siRNA mixture.
4.
Input the electroporation parameters in the Input window and perform
electroporation using the parameters listed below.
Results
Sample Well
no. Pulse
Voltage
Pulse
Width
Pulse
no.
Transfection Efficiency Cell Viability
1
A1
Use pre-optimized parameter or control without electroporation.
2 A2 2000 20 1
3 A3 2050 20 1
4 A4 2100 20 1
5 A5 2150 20 1
6 A6 2200 20 1
7 B1 2250 20 1
8 B2 2300 20 1
9 B3 2350 20 1
10 B4 2400 15 1
11 B5 2450 15 1
12 B6 2500 15 1
13 C1 2000 15 2
14 C2 2050 15 2
15 C3 2100 15 2
16 C4 2150 15 2
17 C5 2200 15 2
18 C6 2250 15 2
5.
After electroporation, immediately remove the Neon
™
Pipette and transfer
samples from the 10 μL Neon
™
Tip into prewarmed 0.5 mL culture medium.
6.
Repeat Steps 3–5 for the remaining samples.
7.
Gently rock the plate to assure even distribution of the cells. Incubate the plate
at 37°C in a humidified CO
2
incubator.
8.
Assay samples to determine the transfection efficiency (e.g., fluorescence
microscopy or functional assay) or gene knockdown (for siRNA). Select the
best conditions and proceed to the next day’s experiment, next page.
Continued on next page
26
