Lipofectamine 2000: Protocol Parameters and Comparison With 3000

Short answer: Lipofectamine 2000 is Thermo Fisher Scientific’s proprietary cationic lipid formulation for transfecting nucleic acids (DNA and RNA) into eukaryotic cells. The vendor’s protocol scales the reagent and nucleic acid by culture-plate format, forms complexes in serum-free medium, and positions Lipofectamine 3000 as its first choice for plasmid delivery. All figures below are vendor-reported and were read on 2026-11-08.

For research use only. This page summarises the manufacturer’s published guidance; always follow the manual supplied with your lot and optimise for your own cell line.

Definition and scope

Thermo Fisher describes Lipofectamine 2000 as a proprietary formulation for transfecting nucleic acids into eukaryotic cells, working through cationic lipid-mediated delivery. The protocol page covers plasmid DNA and siRNA or Stealth RNAi oligonucleotides. Complexes can be added to cells in the presence or absence of serum (Thermo Fisher protocol page). It is a reagent-based method, distinct from polymer reagents such as those discussed in our guide to polyethylenimine transfection, and from the newer reagent covered in the Lipofectamine 3000 protocol guide.

Lipofectamine 2000 protocol: how it is used in practice

The vendor’s workflow has the same shape in every plate format. The parameters below come from the protocol page.

• Cell density at transfection: adherent cells for plasmid DNA 70-90% confluent (0.5-2 x 10⁵ cells per well in the 24-well example); suspension cells 4-8 x 10⁵ per well; siRNA transfections 30-50% confluent.
• Antibiotics: the protocol page states not to add antibiotics to the medium during transfection because this causes cell death.
• Complex formation: dilute the nucleic acid in serum-free Opti-MEM I; separately dilute the reagent and hold it for 5 minutes at room temperature; combine and incubate 20 minutes at room temperature. The vendor states the complexes are stable for 6 hours at room temperature.
• After addition: a medium change after 4-6 hours is optional. Plasmid assays are typically run 18-48 hours later; siRNA knockdown is assessed at 24-96 hours.

Reference table: vendor-listed amounts per well

Plate format	Plasmid DNA / reagent	siRNA / reagent	Diluent per component	Plating medium
96-well	0.2 µg / 0.5 µL	5 pmol / 0.25 µL	25 µL	100 µL
24-well	0.8 µg / 2.0 µL	20 pmol / 1.0 µL	50 µL	500 µL
6-well	4.0 µg / 10 µL	100 pmol / 5 µL	250 µL	2 mL

Source: Thermo Fisher Lipofectamine 2000 protocol page, read 2026-11-08. These are starting points from the vendor, not validated settings for any particular cell line.

Lipofectamine 2000 vs 3000

Thermo Fisher’s FAQ calls Lipofectamine 3000 the best option for plasmid DNA across a broad range of cell types and the most gentle, while describing Lipofectamine 2000 as an alternative that may work better for certain cell types. The table sets out what the vendor pages state.

Question	Lipofectamine 2000	Lipofectamine 3000	Source
Vendor’s recommendation for plasmid DNA	Alternative that may suit certain cell types	Best option across broad cell types	Product FAQ
Co-transfecting plasmid and siRNA	Listed as an option	Listed as an option (with the P3000 enhancer)	Product FAQ
17 cancer cell line panel: lines classed “easy to transfect”	25%	60%	Lipofectamine 3000 product page (vendor data)
Protein expression, about 80 lines	Baseline	1- to 17-fold improvement reported	Lipofectamine 3000 product page (vendor data)

The comparison data are the manufacturer’s own; the sources read did not include an independent head-to-head study. For a siRNA-only experiment the FAQ recommends Lipofectamine RNAiMAX, which it says cannot be used to co-transfect siRNA with plasmid (product FAQ).

Where it matters for labs: choosing and storing the reagent

• Which reagent for which cargo (vendor FAQ): plasmid DNA, Lipofectamine 3000 first, with 2000 as an alternative; siRNA or miRNA, RNAiMAX; plasmid plus siRNA together, Lipofectamine 3000 with P3000 or Lipofectamine 2000.
• Storage: the FAQ gives 4 °C and 12 months’ stability and says the reagent should never be frozen.
• Stable lines: the FAQ places selection antibiotics 24-48 hours after transfection.
• Related terms: institutional biosafety committee and biosafety cabinet.

Common misunderstandings: where vendor guidance differs

• Confluence. The protocol page gives 70-90% for adherent plasmid transfections, while the FAQ says the reagent works best in cultures above 90% confluent. Treat the range as something to optimise per cell line rather than a fixed rule.
• Serum. Serum does not have to be absent from the cultures during transfection, but the FAQ says it is critical to form the lipid-nucleic acid complex without serum.
• Antibiotics. Antibiotics are fine for routine culture. The protocol page says not to add them during transfection; the FAQ says not recommended unless previously tested.
• Freezing. The FAQ entries are not fully consistent about an accidental freeze-thaw; the vendor’s advice is not to use reagent that has been frozen.
• Diagnostic use. Thermo Fisher’s product pages carry a “For Research Use Only. Not for use in diagnostic procedures” footer.

FAQ

What is Lipofectamine 2000 used for?

Transfecting plasmid DNA and RNA (siRNA, Stealth RNAi) into eukaryotic cells in research, per the vendor protocol page.

What is the Lipofectamine 2000 protocol?

Dilute nucleic acid and reagent separately in serum-free medium, combine for 20 minutes, add to cells at the vendor-stated density, and assay at 18-48 hours (DNA) or 24-96 hours (siRNA). See the amounts table above.

Is Lipofectamine 2000 better than 3000?

The vendor recommends 3000 first for plasmid DNA and reports higher efficiency in its own panels, while noting 2000 may work better for certain cell types. Test both on your cell line if it matters.

Can serum be present?

Complexes can be added in the presence or absence of serum, but they must be formed without it.

Sources

• Thermo Fisher, Lipofectamine 2000 transfection protocol (read 2026-11-08)
• Thermo Fisher, Lipofectamine 2000 product FAQs (read 2026-11-08)
• Thermo Fisher, Lipofectamine 3000 reagent page (vendor comparison data, read 2026-11-08)