Optimization Protocol for DNA and siRNA,
Continued
24-well
Optimization
Protocol for
Adherent and
Suspension Cell
Lines—Day One
1.
Make sure you have cells prepared as described on pages 16–17, have the DNA
or siRNA, and prepare 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/siRNA for at least 30 transfections.
2.
For each electroporation sample using the
10 μL Neon
™
Tip
in
24-well
format,
see table below. For using the 100 μL Neon
™
Tip in
24-well
format, adjust the
amounts listed in the table below appropriately by 10-fold.
Cell Type
Cell no.
DNA
siRNA
Resuspension
Buffer R
Adherent 1
×
10
5
/well
0.5 μg DNA/well
15 μg/plate
50 pmol in 10 μL tip
100 nM per well
10 μL/well
285 μL/plate
Suspension 2
×
10
5
/well
1 μg DNA/well
30 μg/plate
100 pmol in 10 μL tip
200 nM per well
10 μL/well
270 μL/plate
3.
Set up a Neon
™
Tube with 3 mL Electrolytic Buffer (use Buffer E for 10 μL
Neon
™
Tip and Buffer E2 for 100 μL Neon
™
Tip) into the Neon
™
Pipette
Station containing the cell-DNA/siRNA mixture as described on page 15.
4.
Press
Optimization
and load the optimization protocols to begin
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 1400 20 1
3 A3 1500 20 1
4 A4 1600 20 1
5 A5 1700 20 1
6 A6 1100 30 1
7 B1 1200 30 1
8 B2 1300 30 1
9 B3 1400 30 1
10 B4 1000 40 1
11 B5 1100 40 1
12 B6 1200 40 1
13 C1 1100 20 2
14 C2 1200 20 2
15 C3 1300 20 2
16 C4 1400 20 2
17 C5 850 30 2
18 C6 950 30 2
19 D1 1050 30 2
20 D2 1150 30 2
21 D3 1300 10 3
22 D4 1400 10 3
23 D5 1500 10 3
24 D6 1600 10 3
Continued on next page
24
