What is the difference between Lipofectamine® and Lipofectamine® PLUS™ reagent?

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Answer

Lipofectamine® and the PLUS™ Reagent are utilized for transfection of a wide range of cell types. In the past, Invitrogen™ sold Lipofectamine® with the PLUS™ reagent. This is no longer the case. Generally speaking, one would not use the PLUS™ Reagent by itself. It is used in conjunction with some other reagent, such as Lipofectamine®, Lipofectamine® LTX or in some cases, with Lipofectin®. Lipofectamine® LTX is offered in combination with the PLUS™ reagent in catalog numbers A12621 and 15338100. We do not recommend using the PLUS™ Reagent with Lipofectamine® 2000 as it does not generally help. .

Answer Id: E3035

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ae8c64bd66d35b9eca16b1d46b810464_FAQ

In the case of the 96-well protocol, Lipofectamine® 2000 requires 320 ng DNA, whereas Lipofectamine® PLUS™, or Lipofectamine® LTX requires up to 200 ng DNA. Does this mean that more DNA is needed to transfect efficiently when using Lipofectamine® 2000?

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Answer

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.

Answer Id: E3062

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b91cf32337065af6e169fe7b0587a527_FAQ

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

Can I use the same amount of any lipid reagent for transfection of my particular cell line?

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Answer

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: E3126

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46c1c8d405f800444c08e5ab73e5feb3_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

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

Are lipid transfection reagents fluorescent?

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Answer

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: E4451

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9be33fef55d66f91b23b1bcdf1cd24fa_FAQ

Which lipid transfection reagent do you recommend for my cell line?

Product FAQ

Answer

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: E4502

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525f62db237d4792d2686a30a0c3ed89_FAQ