Life technologies invitrogen Neon, User Manual

Page: 26 / 52

Using the Neon
™
Transfection System,
Continued
Electroporation
Protocol
1. Make sure you have appropriate number of cells prepared as described on
pages 16–17, have the plasmid DNA or siRNA at the suggested concentrations
(page 13), and prepare a plate containing culture medium
without antibiotics
to transfer the electroporated cells.
For details on optimizing the transfection efficiency of your cells, see page 22.
2. For each electroporation sample, the recommended amount of plasmid DNA
or siRNA, cell number, and volume of plating medium
per well are listed
below. Use Resuspension Buffer T for primary suspension blood cells.
Format Cell Type DNA (μg) siRNA
(nM) Neon ™ Tip Vol. plating
medium Cell no. Buffer R or
Buffer T*
Adherent 0.25–0.5 10 μL 1–2 × 10 4 10 μL/well
96-well
Suspension 0.5–1
10–200
10 μL
100 μL
2–5 × 10
4
10 μL/well
Adherent 0.25–1 10 μL 2.5–5 × 10
4
10 μL/well
48-well
Suspension 0.5–2
10-200
10 μL
250 μL
5–12.5 × 10
4
10 μL/well
Adherent 0.5–2 10 μL 0.5–1 × 10
5
10 μL/well
24-well
Suspension 0.5–3
10-200
10 μL
500 μL
1–2.5 × 10 5 10 μL/well
Adherent 0.5–3 10 μL 1–2 × 10
5
10 μL/well
12-well
Suspension 0.5–3
10-200
10 μL
1 mL
2–5 × 10
5
10 μL/well
Adherent 0.5–3
(10 μL)
5–30
(100 μL)
10 μL/100 μL 2–4 × 10
5
10 μL or
100 μL/well
6-well
Suspension 0.5–3
(10 μL)
5–30
(100 μL)
10-200
10 μL/100 μL
2 mL
0.4–1 × 10
6
10 μL or
100 μL/well
Adherent 5–30 100 μL 0.5–1 × 10
6
100 μL/well
60 mm
Suspension 5–30
10-200
100 μL
5 mL
1–2.5 × 10
6
100 μL/well
Adherent 5–30 100 μL 1–2 × 10
6
100 μL/well
10 cm
Suspension 5–30
10-200
100 μL
10 mL
2–5 × 10 6 100 μL/well
*Use Resuspension Buffer T for primary suspension blood cells.
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
(page 15).
4. Set the desired pulse conditions on the device based on your cell type (page 7).
5. Transfer the appropriate amount of plasmid DNA/siRNA into a sterile, 1.5 mL
microcentrifuge tube.
6. Add cells to the tube containing plasmid DNA/siRNA and gently mix. See the
above table for cell concentration, DNA, and plating volumes to use.
7. To insert a Neon
™
Tip into the Neon
™
Pipette, press the push-button on the
pipette to the second stop to open the clamp.
Continued on next page
18