Optimization Protocol for DNA and siRNA,
Continued
Optimization
Protocol—Day Two
Select the best transfection conditions obtained from the previous experiment
and fine-tune the optimization by narrowing the
Pulse Voltage
.
For example, if you obtained optimal conditions between 1,500 V, 20 ms and
1,400 V, 30 ms, (underlined in the table on the next page) perform optimization
using these narrower parameters as below.
1.
Make sure you have cells prepared as described on pages 16–17, have the
DNA or siRNA, and prepare 18- or 24-wells of a 24-wells plate with 0.5 mL
culture medium with serum and
without antibiotics
to transfer the
electroporated cells.
2.
For each electroporation sample using the
10 μL Neon
™
Tip
, 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
Format
Cell no.
DNA
siRNA
Resuspension
Buffer
Adherent 24-well
1
×
10
5
/well
0.5 μg DNA/well
15 μg/plate
50 pmol in 10 μL tip
100 nM per well
Buffer R
10 μL/well
285 μL/plate
Suspension 24-well 2
×
10
5
/well
1 μg DNA/well
30 μg/plate
100 pmol in 10 μL tip
200 nM per well
Buffer R
10 μL/well
270 μL/plate
Primary
Suspension
Blood Cells
18-well
1–2 × 10
5
/well 0.5–1
μg
DNA/well
20 μg/plate
100 pmol in 10 μL tip
200 nM per well
Buffer T
10 μL/well
180 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 and Neon
™
Tip containing the cell-DNA/siRNA mixture.
4.
Perform electroporation using the parameters listed on the next page:
Continued on next page
27
