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MPK10025

Product FAQ

Can I use the Neon electroporation device for RNAi applications?

Answer

Yes. The Neon Transfection System can be used for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type-specific protocol for DNA, or use the pre-programmed 24 step optimization protocol.

Answer Id: E5495

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Product FAQ

What is the advantage of the Neon transfection chamber design over standard cuvettes?

Answer

Unlike standard cuvette-based electroporation chambers, the Neon system uses a biologically compatible pipette tip chamber. The design of a gold coated wire electrode inside a pipette tip has been shown to produce a more uniform electrical field and a lower pH gradient across the cell suspension. Therefore, this design allows for a better maintenance of physiological conditions resulting in very high cell survival compared to conventional electroporation*.

* Kim JA, Cho K, Shin MS, et al. (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23(9):1353-1360.

Answer Id: E5496

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Product FAQ

What plasmid preparation method do you recommend to electroporate with the Neon System?

Answer

We recommend using anion exchange chromatography to prepare transfection grade plasmid DNA. This technology is found in our PureLink HiPure plasmid purification kits. Do not use standard mini-prep spin columns, as they contain silica membranes which do not remove impurities to the same extent as anion exchange resins.

Answer Id: E5497

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Product FAQ

Can the Neon System be used for co-transfecting siRNA and a plasmid construct?

Answer

We have data which shows knockdown of EmGFP by a specific siRNA which was co-transfected with an EmGFP expressing plasmid, as well as knockdown of endogenous genes.

Answer Id: E5520

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Product FAQ

Can the Neon System be used for making stably-transfected cell lines?

Answer

While we would expect this to be possible, we do not have in-house data to support this application.

Answer Id: E5498

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Product FAQ

Can I use the Neon system for co-transfecting two or more plasmids?

Answer

We currently do not have data to support this, but co-transfection of different plasmids should work. However, the amount of DNA should be carefully titrated, since overloading the cells with plasmid DNA or using unfavorable ratios of the plasmids may cause toxicity. Therefore, we recommend starting optimizations of co-transfection experiments with low amounts of DNA followed by a stepwise increase. Various ratios of the plasmids should be tested if toxicity is observed.

Answer Id: E9118

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Product FAQ

Does transfection efficiency vary with the 10 μL and 100 μL Neon tips? Can instrument settings determined for the 10 μL tips be used with the 100 μL tips?

Answer

There is no reason to speculate that an optimized electroporation parameter would be different between 10 μL and 100 μL Neon tips. Therefore, even though the table in the Neon Cell Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html) specifies the 10 μL tip, those conditions should be okay for 100 μL tips, as long as the recommended density of cells is used. If reduced transfection efficiency is observed, fine-tuned adjustment of the voltage settings may be required to improve efficiency. A 24-well optimization with the 100 μL tips is usually not necessary.

Answer Id: E9124

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Product FAQ

Will a low A260/A280 ratio lead to both reduced transfection efficiency and cell viability in the Neon device?

Answer

Yes. To check the quality of your DNA, we strongly recommend determining the A260:A280 ratio. It should be at least 1.6 for a good DNA preparation.

Answer Id: E9004

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Product FAQ

What is the minimum number of cells you can transfect in one reaction with the Neon System?

Answer

We have transfected as few as 30,000 cells using the 10 microliter tips. If the cell density is too low during electroporation, viability will typically be compromised. This effect is somewhat cell type-dependent. Therefore, how low you can go with your cell line or primary cell type without substantially reducing viability needs to be determined empirically. One of our customers has reported successful transfection of 5000 primary hair follicle cells.

Answer Id: E5521

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Product FAQ

Does linearization improve stable integration of my plasmid?

Answer

Circular and linearized plasmids which do not contain special recombination sequences generally transfect with the same efficiency and integrate into the genome with similar probability. However, the area of recombination on the plasmid can be influenced by linearization, as loose ends are preferred over continues stretches of sequence. By linearizing the plasmid, you can thus determine at which position within the plasmid the recombination occurs, thereby conserving the expression cassette in most cases.

Answer Id: E5500

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Product FAQ

Can I use the Neon system for co-transfecting siRNA and a plasmid construct?

Answer

Yes, we have in-house data that show knockdown of EmGFP by a specific siRNA that was co-transfected with an EmGFP-expressing plasmid, as well as knockdown of endogenous genes.

Answer Id: E9119

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Product FAQ

When do you use Neon Buffer T?

Answer

Buffer T is an alternative cell resuspension buffer for use with primary blood cells. Its composition differs from that of Buffer R and allows the application of higher voltages due to lower conductivity. Buffer T works with T-cells, B-cells, monocytes, and PBMCs as well as bone marrow-derived cells. It does not work with established cell lines or primary cells which have been kept in culture for some time. In some situations it is not immediately clear whether Buffer R or Buffer T would work, so both should be tried in separate optimization experiments.

Answer Id: E5528

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Product FAQ

After Neon electroporation, how long should I wait before analyzing protein expression?

Answer

The optimal time point for analysis of protein expression is related to the stability of the protein being expressed. The half-life of protein products can range from less than a few minutes to several days. For a short-lived protein (like luciferase), protein expression analysis should be done at 6-18 hours post-electroporation. For a more stable protein such as GFP, the analysis can be done 24 hours post-electroporation or even a little later.

Answer Id: E9125

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Product FAQ

Does Invitrogen have an optimization service for the Neon System?

Answer

We do not offer such a service at this time. The Neon Transfection System is designed to facilitate the optimization of transfection conditions. Typically, three rounds of optimization are sufficient to find the best instrument settings for any given cell line or primary cell type. Unless you prepare your cells from very small amounts of tissue, or tissue which is difficult to process, optimizing your cells should not take more than a week and costs a lot less than a custom service.

Answer Id: E5487

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Product FAQ

What are the important considerations with Neon transfection of large plasmids?

Answer

For large plasmids, it is important to prepare highly concentrated plasmid. For the control plasmid, 0.5 mg is used for 10 mL electroporation and the size of the control plasmid is ~5.5 kb. Let’s say the large plasmid being used is 50 kb. This is almost 10 times larger than the control, so you would have to use 10 times more plasmid to compensate for the molecular number. So you would use 5 mg in 10 mL electroporation. To cover this large amount of plasmid, the plasmid concentration should be over 5 mg/mL. One thing to keep in mind is that when you add large amounts of plasmid, it can damage cells due to toxicity of the plasmid sample itself. Thus, we recommend optimizing, starting with a plasmid amount that is less than the amount you came up with, and then moving up in plasmid amount while checking both viability and transfection efficiency.

Answer Id: E9001

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