Are there any guidelines for choosing a lipid transfection reagent?

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Answer

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

Answer Id: E3079

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a44e61a1d926f9486ec0d15596251cba_FAQ

What kind of tubes can I use to form DNA:lipid complexes?

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Answer

We recommend using polypropylene tubes.

Answer Id: E3124

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aab9bac642dd798e1a1bf44d4408dc76_FAQ

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?

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Answer

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: E3125

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1c1f5027dd54726203418309d0a5000b_FAQ

Does the method of generating lipid-DNA complex affect transfection efficiency?

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Answer

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

Answer Id: E3127

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b85873045a0a02ad90bfd8aea5853fb9_FAQ

What types of molecules can be transfected with cationic lipid reagents?

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Answer

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: E3128

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25a97d644624328d60fc3a57921caf82_FAQ

Can antibiotics be used in media during transfection?

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Answer

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: E3129

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b696afc7c66bb4ee77c91bbe14d9bc49_FAQ

Is it necessary to use serum-free media during lipid transfection?

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Answer

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: E3131

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d128a808b58012e27949dbd6ad773aab_FAQ

What is the shelf life of the lipid transfection reagents?

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Answer

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: E3132

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28e9e45a965960f6bf96fdfd9b2dadf5_FAQ

Can lipid reagents be used to cotransfect plasmids?

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Answer

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: E3133

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e511becc41e99f20519fe84d901b4e66_FAQ

How do cationic lipids compare with calcium phosphate in transfection efficiency?

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Answer

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: E3137

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dec9a0f002f2e91166d3ae74df29452e_FAQ

How do I scale up or down my transfection reaction?

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Answer

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: E3138

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e17c91a9c4d7125012544e0f649519ca_FAQ

Do I really need to include a control when doing siRNA transfection?

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Answer

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: E3850

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909cf4cdab2e47d21af7eb22a6ab0f97_FAQ

Is the passage number of my cells important to consider when doing transfection?

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Answer

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: E3852

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b3c877b15a236b74a2be3e3809141d4d_FAQ

How can I improve transfection efficiency with cationic lipids?

Product FAQ

Answer

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: E3994

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aa1760ce6c7e07c9f4e5159cd4ac6660_FAQ

Why do I see precipitates on the cells after transfection?

Product FAQ

Answer

A small granular-like precipitate may be detected microscopically on the cells after transfection using cationic lipid. This is normal. The presence or absence of this precipitate is not indicative of the transfection efficiency. The precipitate can be caused by presence of excess EDTA or cationic lipid. Use DNA in water or, if in TE, use EDTA concentrations of <0.3 mM in the diluted DNA. Ensure concentrations of cationic lipid reagents do not exceed recommended amounts in complex formation.

Answer Id: E3996

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eff0fb47779ff7e7d978202b29f0cdba_FAQ