Optimization Protocol for DNA and siRNA,
Continued
Optional:
Optimization
Protocol—Day
Three
For further optimization, repeat experiments by varying other conditions such
as multiple pulsations.
This is optional and depends on the cell type.
For siRNA, you can vary the amount of siRNA from 10–200 nM.
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-well plate containing
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
29
