I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

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Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.

Answer Id: E8964

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Why is my transfection not reproducible?

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In general, transfection efficiency will show some degree of variability from one transfection to another, no matter how hard one tries to control the parameters of transfection. Keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. To minimize the effect of transfection variability, one can use an internal reference control such as beta-galactosidase or luciferase. Co-transfect the expression plasmid with the reference plasmid and assay for the activity of beta-gal or luciferase.

Answer Id: E8965

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Why would the expression level of my gene in transient transfectants be greater than that in stable transfectants?

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Expression in transiently transfected clones is typically higher because transiently transfected cells have a higher copy number of the gene (hundreds per cell). Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Answer Id: E8966

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Where can I find cell line-specific transfection protocols?

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Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the protocol does not perform as expected, we recommend testing the conditions described in the protocol supplied with the product to determine the optimal protocol. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result.

Answer Id: E8967

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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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Is there a place where I can find references from other researchers who have used your reagents?

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Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Answer Id: E8968

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What types of molecules can be transfected using your transfection reagents?

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Our cationic lipid transfection reagents can be used to transfect DNA, siRNA, Stealth RNAi™ siRNAs, mRNA, Dicer-generated siRNA pools, or plasmids containing shRNA cassettes. Oligonucleotides, proteins, and RNA can also be transfected. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb.

Answer Id: E8986

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Do mammalian cells in culture divide in the presence of G418?

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Yes, cells can continue to divide up to two times in the presence of lethal doses of G418. The effect of the drug usually becomes apparent by two days.

Answer Id: E8969

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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. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.
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 may contain components that could interfere with complex formation. We recommend using Opti-MEM™ I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, keeping in mind that the efficiency of complex formation may not be as high as with Opti-MEM™ I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, 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% confluency at the time of transfection (for Lipofectamine™ 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase. For better consistency of results between transfection experiments, it would be best to accurately count your cells with a hemocytometer or with the Countess™ II FL Automated Cell Counter (Cat. No. AMQAF1000).
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 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

Also, please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Answer Id: E3994

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I accidentallly left my lipid reagent at room temperature. Can I still use it?

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Answer

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Answer Id: E9065

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Can I use Lipofectamine™ 3000 in RNAi experiments?

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Answer

Lipofectamine™ 3000 works well for delivery of vector-based RNAi and synthetic siRNA, as well as co-transfection of siRNA with plasmid DNA. However, Lipofectamine™ RNAiMAX is our best transfection reagent for synthetic siRNA. For in vivo siRNA experiments, we recommend Invivofectamine™ 2.0 Reagent.

Answer Id: E9016

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What is the stability of your transfection reagents?

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Our transfection reagents are shipped under ambient conditions and should be stored at 4 degrees C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of receipt unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance. Please see this white paper (http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf) on ambient shipping of Lipofectamine™ transfection reagents.

Answer Id: E8988

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What are the different methods available for transfection?

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Answer

A wide variety of gene delivery techniques are available to introduce plasmid DNA, siRNA or duplex RNAi, oligonucleotides, and RNA into eukaryotic cells for a variety of research and drug discovery applications. A review of these techniques with pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Answer Id: E8960

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Can I use Lipofectamine™ LTX in RNAi experiments?

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Answer

Lipofectamine™ LTX works well for delivery of vector-based RNAi but for direct transfection of synthetic siRNA, we recommend using Lipofectamine™ RNAiMAX. For in vivo siRNA experiments, we recommend Invivofectamine™ 2.0 Reagent.

Answer Id: E9025

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Can antibiotics be used in the medium during transfection?

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Answer

Yes, antibiotics (penicillin-streptomycin) can be used in the medium during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For certain cell types that are sensitive to transfection or have toxicity issues, omitting antibiotics may improve results, however. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Answer Id: E8998

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