General Guidelines,
Continued
DNA Quality and
Amount
The quality and concentration of DNA used for electroporation plays an
important role for the transfection efficiency. We strongly recommend using
high quality plasmid purification kits such as PureLink
™
HiPure Plasmid DNA
Purification Kits (page 38) to prepare DNA.
Resuspend the purified DNA in deionized water or TE buffer (10 mM Tris-
HCl, 1 mM EDTA, pH 8.0) at a concentration between 1–5 μg/μL.
Concentrations may vary depending on cell type.
The DNA amount should not exceed 10% of total volume used.
Check the purity of the purified DNA preparation by measurement of the
A
260/280
ratio. The ratio should be at least 1.8 for electroporation.
The device has been routinely tested with 4–7 kb plasmids and plasmids up
to approximately 20 kb should not be a problem. Using plasmids larger than
20 kb will most likely lower transfection efficiency.
Important
Do not precipitate DNA with ethanol to concentrate DNA. Concentrated DNA
by ethanol precipitation shows poor transfection efficiency and cell viability due
to salt contamination.
siRNA Quality and
Amount
The quality and concentration of siRNA used for electroporation plays an
important role for the transfection efficiency. We strongly recommend using
high quality siRNA such as Stealth
™
,
Silencer
®
Select, or
Silencer
®
siRNA.
The recommended starting siRNA concentration is 100–250 μM in nuclease-
free water.
The siRNA amount should not exceed 10% of total volume used.
Controls
GFP Control
To initially assess transfection efficiency for your cell type using fluorescent
microscopy, we recommend using a plasmid encoding GFP (green fluorescent
protein) or any colored variant of GFP (Clontech or equivalent). For best
results, the vector encoding the GFP should have the following features:
Strong promoter active in a variety of mammalian cells such as the
immediate early CMV (cytomegalovirus) promoter
SV40 polyadenylation signals downstream of the GFP gene for proper
processing of the 3' end of the GFP mRNA.
Antibiotic selection marker
pUC origin of replication for propagation in
E. coli
siRNA Control
For siRNA experiments, use BLOCK-iT
™
Fluorescent Oligo
for electroporation
or
Silencer
®
Select GAPDH Positive Control siRNA (page 38) to assess
transfection efficiency.
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