What kind of tubes can I use to form DNA:lipid complexes?
I have optimized my conditions for a particular cell line but I seem to be getting inconsistent results with my transfection. What factors contribute to this inconsistency?
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.
Answer Id: 3125
Does the method of generating lipid-DNA complex affect transfection efficiency?
What types of molecules can be transfected with cationic lipid reagents?
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).
Answer Id: 3128
Can antibiotics be used in media during transfection?
We discourage using any antibiotics during transfection (e.g. Geneticin®, Hygromycin, Gentamycin, Penicillin, etc). There can be higher cell death when antibiotics are present during transfection. Even though some such as penicillin and streptomycin are not toxic to eukaryotic cells in a healthy culture, during transfection the cell permeability increases so that much higher levels of antibiotics get into cells. For stable transfections, wait at least 24-48 h after transfection before adding selective antibiotics.
Answer Id: 3129
Is it necessary to use serum-free media during lipid transfection?
Not in all cases. What is essential is to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum containing medium. For optimal results, with Lipofectin® perform transfection in medium without serum.
Answer Id: 3131
What is the shelf life of the lipid transfection reagents?
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.
Answer Id: 3132
Can lipid reagents be used to cotransfect plasmids?
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.
Answer Id: 3133
How do cationic lipids compare with calcium phosphate in transfection efficiency?
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.
Answer Id: 3137
How do I scale up or down my transfection reaction?
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
Answer Id: 3138
Are lipid transfection reagents fluorescent?
The intrinsic fluorescence of Lipofectamine® 2000 and Lipofectamine® under FITC conditions was examined and the results were as follows:
- Lipofectamine® 2000 in serum-free conditions (with or without DNA) - weak whitish fluorescence.
- Lipofectamine® 2000 in serum-containing conditions (with or without DNA) - medium white and red fluorescence.
- Lipofectamine® & Lipofectamine® PLUS (with or without DNA) - orange fluorescence.
- We haven't seen any significant green fluorescence attributable to the Lipofectamine®.
These observations were made after transfection of CHO-K1 cells and by examining the live cells in PBS. Other lipid transfection reagents were not studied extensively on this issue, but it is very likely they will produce fluorescence in transfected cells.
Fluorescence from various media components may be where the problem lies. For example, riboflavin apparently fluoresces in the green wavelength and thus DMEM, which has a high riboflavin content, can be problematic in fluorescence experiments. Ham's F12 medium is 10x lower in riboflavin and is more suitable for fluorescence work.
One possible solution is to perform fluorescence imaging in PBS, rather than in culture medium. Also, check to make sure the cells are healthy and intact. Lysed cells or cells under stress may generate autofluorescent products.
Answer Id: 4451
Which lipid transfection reagent do you recommend for my cell line?
The best transfecting agent and efficiency would depend on the particular cell line you have. Please visit our online selection tools to see our recommendation for your specific cell line. If your cell line is not on the list, we recommend you try Lipofectamine® LTX or Lipofectamine® 2000 for plasmid transfection, and Lipofectamine® RNAiMAX for siRNA transfection. For primary cells, Lipofectamine® LTX with PLUS™ reagent is generally the best choice for plasmid transfection. For some hard-to-transfect cells, like suspension cells and stem cells, the Neon™ electroporation system usually works better compared to lipid transfection reagent.
Answer Id: 4502
Do I really need to include a control when doing siRNA transfection?
It is absolutely critical to have a control oligonucleotide to be able to determine any non-specific effects. This oligonucleotide can be a “scrambled” oligonucleotide (same length and base composition in a random order) or a “sense” oligonucleotide if the target is mRNA.
Answer Id: 3850
Is the passage number of my cells important to consider when doing transfection?
In general, once optimal transfection conditions are determined for a given cell line, it is recommended that cells be passaged less than 20 times to maintain reproducible results. Thus immediately following the determination of optimal conditions, cells should be frozen down so that when the working stock approaches 20 passages, a new batch can be started from the frozen stock.
Answer Id: 3852
How can I improve transfection efficiency with cationic lipids?
1. Select the cationic lipid reagent that is likely to result in highest transfection efficiency for your cell type. See the references listed in the Cell Lines database on our web site.
2. Optimize the cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum contains lipids which will interfere with complex formation. Opti-MEM® I Reduced-Serun Medium or DMEM (use either medium in the absence of serum during complex formation) is good media for complex formation.
4. Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be from 50% to 80% confluent at the time of transfection (for Lipofectamine® 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase.
6. Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is below 4 degree.
8. Include a positive control for the transfection assay.
Answer Id: 3994