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

Are lipid transfection reagents fluorescent?

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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?

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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

Why do I have to dilute my DNA in medium before adding the PLUS™ reagent? Can I just add them together?

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Answer

No, it is important to dilute the DNA into medium and mix before adding the PLUS™ reagent or the DNA may precipitate.

Answer Id: E3849

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b427970680580ac1afa6cc01e6514e30_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?

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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?

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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

Is my lipid (Lipofectin®, Lipofectamine®, DMRIE-C, Lipofectamine® 2000, Oligofectamine™, Cellfectin® II) supposed to be cloudy?

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Answer

It is normal to see some turbidity and cloudiness in DMRIE-C, Cellfectin® II and Lipofectamine® 2000, and the others should be clear. Lipid transfection reagents are sensitive to low temperature; if you put them at temperature below 4 °C they will precipitate or even freeze and lower the efficiency. Most lipids will go cloudy and precipitate upon freezing, and may not be active anymore.

Answer Id: E4000

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7e70e5b8ab3b4ffb0b5b632ee6ab89f2_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?

Product FAQ

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