Lipofectamine 3000 Protocol: Per-Well Amounts, Steps, and siRNA Differences

The Lipofectamine 3000 protocol in Thermo Fisher’s user guide (publication MAN0009872) is a three-part procedure: plate cells to 70-90% confluence, mix plasmid DNA with P3000 Reagent and Lipofectamine 3000 Reagent in Opti-MEM to form complexes, then add the complexes to the cells. The manual recommends testing two Lipofectamine 3000 amounts the first time. This page puts the per-well amounts for 96-, 24- and 6-well plates in one table, shows the arithmetic, and explains the one change needed for siRNA. Lipofectamine 3000 is labelled for research use only, not for use in diagnostic procedures.

The page covers the plasmid DNA workflow and the siRNA variant. For choosing a transfection method and the approvals around it, see transfection methods, controls and biosafety approval; for the general chemistry of lipid delivery, see lipofection.

What you need

Thermo Fisher describes Lipofectamine 3000 as a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells, designed especially for hard-to-transfect cells. According to the user guide, a plasmid DNA transfection needs:

• Lipofectamine 3000 Reagent and the P3000 Reagent supplied with it (store at 4 °C; do not freeze).
• Plasmid DNA, with a stock concentration of 0.5-5 µg/µL.
• Opti-MEM Reduced Serum Medium, used to dilute the DNA and the lipid.
• Microcentrifuge tubes and the culture plates you are transfecting.

The cover page of the guide gives the timing as about 10 minutes of preparation, a 10-15 minute complex incubation, and a final incubation of 1-3 days before analysis.

Per-well amounts for 96-, 24- and 6-well plates

The manual lists amounts per well and states that volumes are scaled proportionally for other plate formats. It recommends two Lipofectamine 3000 amounts for any new transfection, because the best amount varies.

Per well	96-well	24-well	6-well
Adherent cells to seed	1-4 × 104	0.5-2 × 105	0.25-1 × 106
Plasmid DNA	100 ng	500 ng	2500 ng
P3000 Reagent	0.2 µL	1 µL	5 µL
Lipofectamine 3000 (low dose / high dose)	0.15 µL / 0.3 µL	0.75 µL / 1.5 µL	3.75 µL / 7.5 µL
Lipofectamine 3000 diluted in Opti-MEM (per tube, two tubes)	5 µL	25 µL	125 µL
DNA master mix in Opti-MEM (total for both doses, split between the two lipid tubes)	10 µL	50 µL	250 µL
DNA in the master mix (total for both doses, 2 x the per-well amount)	0.2 µg	1 µg	5 µg
P3000 Reagent in the master mix (total for both doses)	0.4 µL	2 µL	10 µL
Diluted DNA added to each lipid tube (half of the master mix)	5 µL	25 µL	125 µL
Complex added to each well	10 µL	50 µL	250 µL

Source: Thermo Fisher Lipofectamine 3000 Reagent user guide, MAN0009872 Rev C.0, checked 2026-10-29.

Checking the arithmetic

Three relationships in the table make it easy to scale to any format. First, P3000 Reagent is 2 µL per µg of DNA: 100 ng gives 0.2 µL, 500 ng gives 1 µL and 2500 ng gives 5 µL. Second, the diluted DNA and the diluted lipid are combined 1:1, and the complex volume added per well (10, 50 or 250 µL) is twice the Opti-MEM volume in one lipid tube (5, 25 or 125 µL). Third, the high lipid dose is exactly twice the low dose in every column (0.15 and 0.3, 0.75 and 1.5, 3.75 and 7.5 µL), which is what the manual means by testing two concentrations.

Steps

The guide’s outline: A, plate the cells; B, prepare the DNA-lipid complexes (two lipid doses); C, add the complexes to the cells. Cells should be 70-90% confluent at transfection, and the table timeline seeds on day 0 and transfects on day 1.

1. Seed adherent cells so they are 70-90% confluent at the time of transfection, using the seeding range for your plate format above.
2. In two tubes, dilute Lipofectamine 3000 Reagent in Opti-MEM (5, 25 or 125 µL per tube for 96-, 24- or 6-well plates) and vortex for 2-3 seconds. One tube gets the low dose and one the high dose.
3. Prepare one DNA master mix in Opti-MEM for both doses (10, 50 or 250 µL for 96-, 24- or 6-well plates) containing 0.2, 1 or 5 µg of DNA, which is twice the per-well amount because it feeds two tubes. Add P3000 Reagent at 2 µL per µg of DNA (0.4, 2 or 10 µL) and mix.
4. Split the diluted DNA (with P3000) between the two lipid tubes: add 5, 25 or 125 µL to each tube of diluted Lipofectamine 3000, 1:1. The master mix is shared, so each tube receives half of it.
5. Incubate 10-15 minutes at room temperature.
6. Add the DNA-lipid complex to the cells (10, 50 or 250 µL per well).
7. Incubate the cells at 37 °C, then analyse. The cover page says 1-3 days; the step table says 2-4 days. The two ranges overlap at 2-3 days, so a first experiment can be read in that window.

Complexes are made in serum-free medium (Opti-MEM) and added directly to cells in their normal medium, with or without serum or antibiotics. According to the guide, it is not necessary to remove the complexes or change the medium after transfection. After transfection of a reporter construct, see luciferase reporter assay for a common readout.

Lipofectamine 3000 protocol for siRNA (RNAi)

For siRNA, follow the plasmid protocol but do not add P3000 Reagent when diluting the siRNA in step 3. Everything else, including the two-dose test and the 1:1 combination with diluted lipid, stays the same. For knockdown design and validation, see shRNA knockdown mechanism, design and validation.

What can go wrong

The user guide does not include a troubleshooting section; these are the points it states that most affect a run.

• Using one reagent amount. The manual says the amount varies and tells you to start every new transfection by testing the two recommended concentrations.
• Cell density outside 70-90% confluence. This is the window the protocol specifies at the time of transfection.
• Freezing the reagents. Storage is at 4 °C; do not freeze.
• Adding P3000 to siRNA. The siRNA variant omits it.
• Working from an old copy. The amounts on this page come from revision C.0 of the user guide (10 February 2016). Check the current revision of the manual before running a protocol.

When to get professional help

For cell-line-specific conditions, Thermo Fisher links protocols and a transfection selection tool from the Lipofectamine 3000 product page, and its technical support is the route for reagent-specific problems. Before transfecting, check with your institution’s biosafety officer or committee: whether a protocol needs approval depends on the nucleic acid and the cells, as covered in transfection methods, controls and biosafety. Viral delivery is a separate workflow; see lentiviral transduction protocol.

FAQ

Is there a Lipofectamine 3000 protocol PDF?

Yes. Thermo Fisher publishes the Lipofectamine 3000 Reagent user guide (MAN0009872) as a PDF, linked in the sources below.

Can I use Lipofectamine 3000 for siRNA?

Yes. Follow the DNA protocol but do not add P3000 Reagent when diluting the siRNA.

Do I need to remove serum or antibiotics?

No. The complexes are added to cells in medium, in the presence or absence of serum or antibiotics.

Do I need to change the medium after transfection?

The manual says it is not necessary to remove the complexes or change or add medium after transfection.

How much Lipofectamine 3000 per well?

For plasmid DNA, 0.15 and 0.3 µL (96-well), 0.75 and 1.5 µL (24-well), or 3.75 and 7.5 µL (6-well), tested as two doses; see the table above.

Can Lipofectamine 3000 be used for diagnostic work?

No. It is labelled for research use only, not for use in diagnostic procedures.

Sources

• Thermo Fisher Scientific, Lipofectamine 3000 Reagent Protocol, publication MAN0009872, Rev C.0 (10 February 2016): documents.thermofisher.com. Checked 2026-10-29.
• Thermo Fisher Scientific, Lipofectamine 3000 Transfection Reagent product page: thermofisher.com. Checked 2026-10-29.