11668-027 - FAQs

It generally yields better transfection activity (as measured by protein expression) than all other lipids in a majority of the cell lines tested. It includes a streamlined, simple protocol where the complexes are added directly to cells without changing media. This lends itself to high throughput applications. It works very well in the presence or absence of serum. Examples of cells that show the highest transfection efficiency with Lipofectamine 2000 include 293 F, 293 H, BE(2)C, CHO-K1, CHO-S, COS-1, COS7-L, Human Primary Fibroblasts, Ht-29, HT-1080, MDCK, MRC-5, PC12 and Vero.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

It is safest not to use it, however if the lipid is thawed very slowly at 4°C and its appearance did not change, it is probably worth trying. Lipofectamine 2000 has survived one freeze/thaw without loss of activity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

The number of cells, DNA, lipid, and medium volumes should be scaled up proportionately to the surface area of the plate. For commonly used culture vessels, please refer to the information below regarding actual area and area relative to a 24-well plate well.

Vessel type, Area (cm2), Area Relative to 24-well
96-well, 0.3 cm2, 0.2
48-well, 0.7cm2, 0.4
24-well, 2 cm2, 1
12-well, 4 cm2, 2
6-well, 10 cm2, 5
35 mm, 10 cm2, 5
60 mm, 20 cm2, 10
100 mm, 60 cm2, 30
150 mm, 140 cm2, 70
T25, 25 cm2, 12.5
T75, 75 cm2, 37.5
T150, 150 cm2, 75
T162, 162 cm2, 81
T165, 165 cm2, 82.5
40-50 ml, 25 cm2, 12.5
250-300 ml, 75 cm2, 37.5
650-750 ml, 162-175 cm2, 81-87.5
900 ml, 225 cm2, 112.5


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

For many cell types, higher efficiencies are observed with cationic lipids than with calcium phosphate. Also, cationic lipid data are more reproducible from experiment to experiment. Calcium phosphate is inexpensive however, but pH variation as little as 0.2 can reduce transfection efficiency significantly.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 ug plasmid, use 0.5 ug of each of two cotransfecting plasmids, or 0.25 ug of each of 4, etc. When performing cotransfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Stored at 4°C in a sealed container, the lipids are stable for 12 months. Do not freeze the lipids. At 4°C with long term storage, due to evaporation concentration of lipid may vary, please briefly spin the contents before use. Add less lipid if you start noticing toxicity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Lipid reagents can be used to transfect DNA, RNA, oligonucleotides and siRNA. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. Lipofectin and Lipofectamine 2000 Reagent have also been used to transfect cells with proteins (Beta galactosidase and T3 polymerase).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

YES. Please follow the recommended procedure for each one of the reagents, found in the product manual.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

No. The amount of lipid for each lipid reagent should be optimized for each cell line. Each lipid reagent has different composition/formulation, which will have different impact on each cell line. Therefore please optimize whenever you have a new lipid for each cell line.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Cells from a different passage number may behave differently. Also, if cells were sitting at confluence prior to plating for transfection, they may not transfect efficiently. To minimize such inconsistencies, passage the cells while they are still growing exponentially. Actively dividing cells transfect better.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

It is best to optimize for your cells and application. Here are some basic guidelines:

- Lipofectamine LTX and PLUS Reagent: Minimal optimization, excellent efficiency with adherent eukaryotic cells DNA, difficult cell lines
- Lipofectamine Reagent: Adherent eukaryotic cells, DNA, oligonucleotides
- Lipofectin Reagent: Transfecting DNA in eukaryotic cell
- Cellfectin II: Insect cells
- DMRIE-C Reagent: DNA, RNA, suspension cells
- Oligofectamine: Oligonucleotides
- Lipofectamine RNAiMAX: siRNA, pre-miR, miRNA, anti-miR

NOTE: Please also visit our online Transfection Selection Tool to get specific recommendation for your cell line


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Yes, you do need more DNA per well when using Lipofectamine 2000, because the optimal confluency of the cells is 90% (as compared to 50-80% confluent with Lipofectamine, or Lipofectamine LTX. Therefore you need more DNA in order to ensure that the maximum number of cells are transfected. With Lipofectamine 2000, we have found empirically that the higher confluence (~90%) of cells at the start of transfection improves transfection efficiency.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.