Neon™ NxT Electroporation System 10-μL Kit, 96 x 2 reactions - FAQs

We recommend warming the buffers to room temperature before use.

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We recommend storing Neon NxT buffers in the original containers at 4 degrees C.

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Neon NxT buffers should be stored at 4 degrees C. Temperature fluctuations and storage at room temperature have been shown to increase the chances for precipitate formation. Therefore, the desired amount of buffer for an experiment should be removed from the buffer bottle and the bottle should be immediately returned to 4 degrees C.

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For best results with the Neon Nxt Electoporation System, the cells should be healthy (not stressed) and actively dividing so that the nuclear membrane integrity is already weakened for payload needing to enter the nucleus. Consider “starving” the cells of serum or other essential nutrients.

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Arcing could be caused by high voltage or pulse length settings, high salt or contaminants in the DNA sample, incorrect cell density, and air bubbles formed during mixing of cell samples. Mix samples gently to avoid air bubble formation and pipette samples in a slow, smooth motion to avoid air uptake.

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The optimal time for analysis of protein expression is dependent upon the stability of the protein being expressed. For a short-lived protein, like luciferase, analysis of protein expression should be done at 6-18 hours after electroporation. For a more stable protein, such as GFP, you should start the analysis at 24 hours or longer post-electroporation.

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We recommend analyzing the cells 72 hours post-electroporation.

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The benefits of the Neon NxT Electroporation System over other electroporation systems are listed below:

- Small to large number of cells can be used. The transfection is performed using as few as 2 X 10E4 or as many as 10 X 10E6 cells per reaction using a sample volume of 10 µL or 100 µL.
- The Neon NxT Electroporation System uses a single transfection kit (Neon NxT Kit) that is compatible with various mammalian cell types, including primary and stem cells, thereby avoiding the need to determine an optimal buffer for each cell type. The two NxT resuspension buffers cover all cell types and applications: Neon NxT Resuspension T Buffer for high voltage protocols >1900 V (naive T cells), and Neon NxT Resuspension R Buffer for all other protocols.
- Open and transparent protocols that are optimized for ease of use and simplicity. TransfectionSelect Protocols & Citations Library (thermofisher.com/transfectionprotocolsandcitations) contains optimized protocols for many commonly-used cell types.
- The Neon NxT Device is preprogrammed with 24-well optimization protocols to optimize conditions for all payloads and cell types.
- The Neon NxT Electroporation System leverages Neon technology that produces 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.

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Yes. However, we recommend waiting for about 4-6 hours before adding antibiotics back to the cells. This is to help ensure that the membrane integrity has been restored.

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Monocytes/macrophages can get activated upon transfection with the Neon NxT Electroporation System, and there are several possible reasons for this:

Monocytes/macrophages respond to very low levels of endotoxin (LPS), which could have been introduced with your plasmid DNA. Make sure that 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 monocyte/macrophage 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 monocyte/macrophage 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 monocyte/macrophage activation. Please make sure that none of these components activates your cells.

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

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

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The most common causes for low cell survival with the Neon NxT Electroporation Device are listed below:
- Sub-optimal electrical parameters
- Poor plasmid quality such as endotoxin contamination
- Plasmid preparation contains high salt concentration
- Plasmid quantity is too high
- Cells are stressed or damaged
- Multiple uses of the same Neon NxT tip

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We do not recommend using the Neon NxT Tip more than twice. The reason for this recommendation is that the wire electrode inside the tip is coated with 24 karat gold. Some of the gold is released each time an electrical pulse is delivered. Therefore, repeated use of the tip will result in the gold coating becoming thin and causing a change in the conductivity of the tip. To be absoutely confident that the correct voltage and current are delivered to your cells, we don't recommend using the tip more than twice.

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In most cases, the Neon/Neon NxT protocol that was determined for the 10 µL tips can be used with the 100 µL tips, but in some cases, reduced transfection efficiency may be observed and adjustment of voltage settings may be required to improve transfection efficiency.

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Potential causes for low transfection efficiency with the Neon NxT Electroporation System include the following:
- Sub-optimal electrical parameters
- Plasmid preparation contains high salt concentration
- Plasmid size is larger than 10 kb
- Plasmid concentration is too low
- Cells are stressed, damaged, or contaminated by mycoplasma
- Cell density is too low or too high
- Cells have undergone high number of passages
- Multiple uses of the same Neon NxT tip

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Cells are analyzed using the Attune NxT Flow Cytometer. Cells are stained with SYTOX Red (1:1,000 dilution) to check viability. Transfection efficiency is measured by GFP-positive cell gating when GFP plasmid is used as a payload.

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The Neon NxT Tubes are disposable and we recommend changing the tubes when changing cells in order to avoid cross-contamination. With regard to plasmid DNA, the answer depends on the sensitivity of your downstream assay. If slight DNA cross-contamination does not affect your assay, you may not have to change the tubes. Please perform pilot experiments to determine whether it works for you. However, we still recommend changing the Electrolytic Buffer. If you run out of Neon NxT Tubes, they can be purchased separately (Cat. No. NT96).

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If you find another cell type in the Neon Nxt Device cell-specific protocols that is similar to your cell type in terms of tissue origin, you can use the electroporation parameters of that cell type for your cell type. You may not get optimal results, but it is a good starting point. For example, say you have 293T cells and you find the HEK293 protocol, since both 293T cells and HEK293 cells are derived from human embryonic kidney, you can use the electroporation parameters of HEK293 cells for 293T cells.

Alternatively, you can use the preprogrammed 24-well optimization protocols in the Neon NxT Electroporation Device to optimize for your cells.

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Unlike standard cuvette-based electroporation chambers, the Neon NxT Electroporation System leverages the proven Neon technology where electroporation takes place in a biologically compatible pipette tip chamber. The design of a gold-coated wire electrode inside of the 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 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).

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We routinely use plasmids of 4-7 kb in our laboratories, and plasmids up to approximately 20 kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. We have some preliminary results indicating that very large BAC DNA can be transfected as well, albeit with a low transfection efficiency.

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The main concern for multiple use of the Neon NxT Tube with Electrolytic Buffer is the possibility of cross-contamination. In addition, we strongly recommend that a new Neon Tube with Electrolytic Buffer be used for each different plasmid DNA/siRNA or cell type.

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Yes. The Neon NxT Electroporation System can be used for electroporation of 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-well optimization protocols included in the Neon NxT Device.

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All buffer compositions are proprietary.

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The Neon NxT Electrolytic E10 Buffer has higher osmolarity than Neon NxT Electrolytic E100 Buffer. The higher osmolarity prevents the leakage of electroporation content from the 100 µL Neon NxT tip which has a larger hole at the tip end than the 10 µL Neon NxT tip.

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Yes. To check the quality of your DNA, we strongly recommend confirming that both A260/A280 and A260/230 ratios are between 1.6 -2.0 and checking for DNA degradation by agarose gel electrophoresis.

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Neon NxT Resuspension Buffer (R or T) is used to resuspend the cells prior to electroporation, whereas Neon NxT Electrolytic Buffer (E10 or E100) is used for electroporation and is added to the Neon NxT Tube prior to electroporation.

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Precipitation may occur if the buffer is not stored at 4 degrees C or if it has been kept at room temperature frequently or for too long. Although precipitation is irreversible, it does not affect the functionality of the buffer. If precipitation appears, we recommend filtering the buffer before use.

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We recommend using Neon NxT Resuspension R Buffer for an electroporation protocol using less than 1900 V while Neon NxT Resuspension T Buffer is recommended for high-voltage electroporation (1900 V and above).

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Neon NxT Resuspension T Buffer is the only buffer in the kit that contains animal-origin components.

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The Neon NxT Electroporation System relies on the same unique and trusted electroporation technology as the Neon Transfection System (Cat. No. MPK5000), but it has new features that make it easier to use. The Neon NxT pulse generator has an improved feedback loop with user interface notification, and ClipTip technology has been incorporated into Neon NxT pipette tips for secure attachment and easy ejection, along with other ergonomic design improvements.

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Neon NxT E10, E100, R, and T buffers have the same compositions as Neon E, E2, R, and T buffers, respectively. However, Neon NxT tips and tubes have different designs compared to the Neon tips and tubes, to improve usability, and therefore are not compatible with the Neon Transfection System.

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