Optimization Protocol for DNA and siRNA
Introduction
Electroporation is mainly dependent on the combination of three electric
parameters such as the electric field, pulse width, and pulse number. Based on
your initial results, you may need to optimize the electroporation parameters
for your cell type especially the hard-to-transfect cells.
The Neon
™
device is preprogrammed with a 24-well optimization protocol
using the 10 μL or 100 μL Neon
™
Tip that allows you to quickly optimize
electric parameters for many adherent and suspension cell lines within days.
For primary blood suspension cells, use the 18-well optimization protocol
with Resuspension Buffer T as described on page 26.
Materials Needed
Ordering information is on page 38.
Neon
™
10 μL or 100 μL Kit
Cells in Resuspension Buffer (prepared as described in pages 16–17)
High quality DNA at a concentration of 1–5 μg/μL in deionized water or
TE buffer or high quality RNAi duplex at a concentration of 100–250 μM in
nuclease-free water (page 13)
Cell culture plates containing the appropriate medium
Workflow
General workflow for optimization is described below. For detailed protocols,
see the next page.
Optimization for plasmid
1.
Perform 24-well optimization using the preprogrammed parameters.
2.
Based on results from Step 1, perform optimization using narrower
(bracket) parameters.
3.
Based on results from Step 2, further refine the parameters to obtain
optimal conditions (this is optional step).
Optimization for siRNA
1.
Perform 24-well optimization using the preprogrammed parameters.
2.
Based on results from Step 1, perform optimization using narrower
(bracket) parameters.
3.
Based on results from Step 2, perform optimization by varying siRNA
final concentrations to 10 nM, 30 nM, 100 nM, and 200 nM.
Continued on next page
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