Transfection Using PEI (Polyethylenimine)
PEI transfection
works through electrostatic interactions between positively charged PEI and
negatively charged DNA, forming PEI-DNA polyplexes. These complexes:
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Attach to the cell membrane,
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Are internalized via
endocytosis, and
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Escape the endosome into the
cytoplasm, where DNA may reach the nucleus.
PEI is widely
used due to its low cost, reproducibility, and ability to transfect many
adherent and suspension cell lines.
Reagents and
Equipment Required
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Mammalian cells
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PEI (25 kDa)
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Plasmid DNA (high purity,
endotoxin-free)
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Serum-free medium
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Complete growth medium (with
serum)
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Tissue culture plates
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CO₂ incubator (37°C, 5% CO₂)
Steps
1. Preparation
of PEI Stock Solution
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Dissolve PEI (25 kDa) in
sterile water at 1 mg/mL.
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Adjust pH to 7.0–7.4 using HCl.
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Filter sterilize (0.22 µm) and
aliquot.
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Store at –20°C. Thaw aliquots
on ice before use.
2. PEI-DNA
Complex Formation
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Dilute 2 µg plasmid DNA in 250
µL serum-free medium in a sterile tube.
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Add 8 dilute 6 µg PEI to the
medium.
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Use a 4:1 PEI:DNA weight ratio.
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Vortex briefly or flick to mix.
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Incubate at room temperature
for 15–20 minutes to allow complex formation.
3.
Transfection
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The cells should be in 70–90% confluency.
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Add the 250 µL PEI-DNA mixture
dropwise to the well.
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Gently swirl the plate to
distribute evenly.
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Incubate at 37°C with 5% CO₂
for 24–72.
4. Analysis
-
Use fluorescence microscopy
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For stable expression, follow
with selection (e.g., antibiotic resistance).