What is the difference between the Neon™ Resuspension and Electrolytic Buffers?

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Answer

The Resuspension Buffer (Buffer R or T) is used to resuspend the cells prior to electroporation, whereas the Electrolytic Buffer (Buffer E or E2) is used for electroporation and is added to the Neon™ tube prior to electroporation.

Answer Id: E9082

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Does linearization improve stable integration of my plasmid?

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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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What is the difference between the Neon™ R and T Resuspension Buffers?

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Both R and T Resuspension Buffers are used to resuspend cells prior to electroporation. Resuspension Buffer R is the standard cell resuspension buffer that is used to resuspend established adherent and suspension cells as well as primary adherent cells. Resuspension Buffer T is an alternative cell resuspension buffer that is used to resuspend primary T and B cells, PBMCs, monocytes, and bone marrow-derived cells. Its composition differs from that of Buffer R and allows the application of higher voltages due to lower conductivity. It does not work with established cell lines or primary cells, which have been kept in culture for some time. In situations where it is not immediately clear whether Buffer R or Buffer T would work, we recommend testing both in separate optimization experiments.

Answer Id: E9083

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I cannot find my cells in your current cell database, will the Neon™ System work with my cells?

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If the cell type has worked with electroporation, it should work with the Neon™ System.

Answer Id: E5502

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Will low A260/A280 ratio of my DNA sample lead to both reduced transfection efficiency and cell viability?

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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: E5538

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How did you select the 24 different instrument settings used to perform a Neon™ optimization?

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These settings were selected based on our experience optimizing numerous cell lines and primary cells in-house. If none of these settings transfect plasmid DNA into your cells, it is unlikely that other conditions will. However, if low transfection efficiencies are obtained with some of the settings, it is likely that they can be further increased by performing additional optimizations to fine-tune your parameters for voltage, pulse width, and number of pulses.

Answer Id: E9108

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What electroporation parameters should I use for siRNA transfection with the Neon™ System if there are no pre-determined parameters?

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Answer

A good start is to use the plasmid electroporation parameters for the same cell type. The Neon™ Cell Database (www.thermofisher.com/neon) contains optimized plasmid electroporation parameters for many commonly used cell types (the list is growing). If the Neon™ Cell Database does not contain the cell type of interest, you can use the 24-well optimization protocol that is preloaded on the Neon device.

Answer Id: E5508

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When should I use Neon™ “T” buffer instead of “R” buffer?

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We recommend using T buffer instead of the standard R buffer for primary blood-derived suspension cells such as primary T and B cells, PBMCs, monocytes, and bone marrow–derived cells. These cells are smaller than regular cell types and require higher voltage for successful electroporation. If you use R buffer and apply high voltage (over 1800 V), you will see sparks or arcing, regardless of cell number and other conditions. The maximum voltage for R buffer is around 1900 V. Buffer T composition differs from that of Buffer R to allow the application of higher voltages due to lower conductivity.

Answer Id: E9084

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How can I use the Neon™ System for cells I would like to transfect that are not found in your current cell database?

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(1) If you find another cell type in the Neon™ Cell Database that is similar to your cell type in terms of tissue origin, you can use the parameters of that cell type for your cell type. You may not get optimal results, but it is a good starting point. One example: your have 293 T cells and you find HEK293 cells in the Neon™ database. Since both are derived from human embryonic kidney, you can use the electroporation parameters of HEK293 cells for 293 T cells.

(2) You can use the preprogrammed 24-well optimization protocol in the Neon™ device to optimize conditions for your cell type

Answer Id: E5503

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Do my monocytes or macrophages get activated upon transfection with your Neon™ system?

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Answer

They could get activated, and there are several possible reasons for this. Monocytes and macrophages respond to very low levels of endotoxin (LPS), which could have been introduced with your plasmid DNA. Make sure you use plasmid DNA which has been purified by anion exchange chromatography, such as our PureLink™ HiPure kits. If you did this and still see activation, perform one or two rounds of PEG precipitation to remove residual endotoxin.

Alternatively, you can subject your plasmid to a second round of anion exchange chromatography purification. If you still get activation, the plasmid itself may contain sequences which stimulate the production of Interferon gamma. It is also possible that certain components in your culture medium, including the FBS batch you are using, may cause activation. Please make sure that none of these components activates your cells.

The procedure for isolating your monocytes is also important. We recommend negative rather than positive selection, as it leaves the monocytes “untouched” by antibodies.

Our electroporation buffers are guaranteed endotoxin-free and do not cause monocyte/macrophage activation in our hands.

Answer Id: E5545

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My Neon™ system R Buffer has a precipitate. Can I put my bottle in a 37°C water bath to dissolve it?

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Answer

No, the precipitate that forms in this buffer is irreversible.

Answer Id: E5539

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Do you offer an optimization service for the Neon™ Transfection System?

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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 little amounts of tissue or tissue that is difficult to process, optimizing the conditions should not take more than a week and would cost a lot less than a custom service would.

Answer Id: E9109

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What optimization is necessary to get the Neon™ System to work?

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Answer

For each cell type in our cell database, we offer thoroughly optimized electrical parameters along with a universal electrolytic buffer. Very little optimization is required. For cells that are not listed in our database, there is a pre-programmed optimization protocol built in the Neon™ device.

Answer Id: E5509

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

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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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When should “T” buffer be used instead of “R” buffer with the Neon™ system?

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Answer

You need T buffer instead of the standard R buffer for primary T and B cells. These cells are smaller than regular cell types and require higher voltage for successful electroporation. If you use R buffer and apply high voltage, you will see sparks.

With R buffer, voltage over 1800V generally generates sparks regardless of cell number and other condition. The maximum voltage for R buffer is around 1900V. In order to apply higher voltage, you need a buffer less conductive than R buffer, which is the T buffer.

Answer Id: E5529

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