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
Can I use the same amount of any lipid reagent for transfection of my particular cell line?
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.
Answer Id: 3126
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
How does the use of Lipofectamine® PLUS™ reagent improve transfection over other cationic lipid reagents?
In general, the transfection activity peak with Lipofectamine® PLUS™ reagent is broader, thus essentially eliminating the need for lengthy optimization experiments. Because the protocol includes a pre-complexing step with PLUS™ reagent, only half the amount of Lipofectamine® reagent is required. The transfection time is short and the transfection can be done in the presence of serum. Difficult to transform cells (e.g. HT29, SKBR3 and MDCK) are transformed at a higher efficiency. See, FOCUS 19.3, 52.
Answer Id: 3163
How do Lipofectamine® PLUS™ and Lipofectamine® compare in transfection efficiency?
Why do I have to dilute my DNA in medium before adding the PLUS™ reagent? Can I just add them together?
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