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Neon™ Transfection System 100 μL Kit, 25 x 2 Reactions - FAQs

View additional product information for Neon™ Transfection System 100 μL Kit - FAQs (MPK10025)

119 product FAQs found

What are the common causes for low cell survival rate using the Neon device?

Here are possible causes for low transfection efficiency using the Neon device:

1. Sub-optimal electrical parameters
2.Poor plasmid quality such as endotoxin contamination
3 .Plasmid preparation containing high salt
4. Plasmid quantity too high
5. Cells are stressed or damaged
6. Using same Neon tip more than two times
7. Microbubbles in tip, causing arcing

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are the common causes for low transfection efficiency using the Neon device?

Here are possible causes for low transfection efficiency using the Neon device:

1. Sub-optimal electrical parameters
2. Plasmid preparation containing high salt
3. Plasmid larger than 10 kb
4. Plasmid concentration too low
5. Cells are stressed, damaged, or contaminated by Mycoplasma
6.Cell density too low or too high
7. Cells with high passage number

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How should I determine siRNA transfection efficiency with the Neon Transfection System?

To determine the Neon transfection efficiency for siRNA, we recommend transfecting the cells with a fluorescent-labeled negative control siRNA (BLOCK-iT Fluorescent Oligo, Cat. No. 13750062) and measuring the transfection efficiency by the percentage of fluorescent-stained cells among viable cells. However, keep in mind that there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescence detection with a microscope does not distinguish the siRNA that enters the cell from the siRNA that sticks to the cell membrane. To measure transfection efficiency more accurately, one needs to transfect the cells with a positive control siRNA such as the one that targets a house-keeping gene, and measure the knockdown of target RNA or protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How should I determine the cell viability and transfection efficiency with the Neon Transfection System?

Cell viability is the number of cells that are confirmed viable from a total cell population. Transfection efficiency is the number of cells that are successfully expressing your construct out of the total number of viable cells (i.e., GFP-positive cells).

Cell viability can be determined by staining cells with propidium iodide or by the trypan blue exclusion method. For adherent cells, cell detachment can be performed using Trypsin or TrypLE Express enzyme prior to staining. Transfection efficiency can be determined using a fluorescence microscope with filter settings appropriate for the detection of GFP (emission: 509 nm). Cells may be counted either by FACS or using the Countess Automated Cell Counter.

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After Neon electroporation, what is the best time point for analysis of siRNA knockdown?

As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA half-life can range from minutes to days (Ross J, 1995, Microbiol Rev 59:423–450) while the half-life of protein products can range from less than a few minutes to several days. In general, the recommended time course ranges from 12 to 72 hours to knock down target mRNA and 24 to 96 hours to adequately knock down target proteins. We recommend measuring mRNA knockdown by qPCR at 8, 24, 48, 72, and 96 hours post-electroporation to determine the time point for maximum knockdown. Also, perform time-course analysis to determine protein knockdown by ELISA (more accurate) or immunoblotting (less accurate).

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

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.

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

Electroporation parameters optimized for the 10 µL tip may be used for the 100 µL Neon tip, but as with any changes in scaling, some optimization may be required. While keeping to the same cell density, we recommend fine-tuned adjustments of the voltage settings to achieve optimal transfection efficiency. A 24-well optimization with the 100 µL tips is usually not necessary.

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Would Neon transfection affect the functional study of membrane proteins?

As with any electroporation method, electrical current is used to create pores in the cell membrane, resulting in some membrane protein damage. To maximize recovery after electroporation, use medium that is optimized for the specific cell type and avoid antibiotics. The recovery time may vary and will depend on cell type and protein. In primary cells, which do not proliferate after electroporation, this membrane damage could be permanent so that it hinders certain membrane protein recovery. Try using lipid transfection first (see Transfection Selection Guide; https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).

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What is the minimum time before antibiotics can be added to the culture after Neon electroporation?

After electroporation, wait for about 4-6 hours before adding antibiotics back to the cells. This is to make sure that the membrane integrity has been restored and to allow adherent cells to attach to the culture vessel.

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What is the maximum number of cells I can transfect in a 100 µL Neon tip?

According to Neon guidelines, you can run up to 5 million cells in 100 µL, this number may vary depending on the size of the cell type.

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What is the minimum amount of cells one can transfect with the Neon system?

As few as 10,000 cells may be used with the 10 µL tips, but this may vary depending on size differences among cell types. One of our customers reported successful transfection of 5,000 primary hair follicle cells. As a general rule, try to avoid using low cell densities as this could reduce viability during electroporation. If it is difficult to avoid low cell densities (ex. primary cells), adjust the voltage to optimize for improved viability. Optimal electroporation conditions are cell type-dependent. Avoid antibiotics in the medium and use medium appropriate for your cell type. For helpful cell-specific electroporation conditions, please visit our Neon cell-specific protocols 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?SID=fr-neon-3

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

Yes, you may co-transfect both plasmid and siRNA together at the same time but some optimization may be necessary.

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

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.

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How do I determine electroporation parameters for my cell line with the Neon Transfection System?

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) offers optimized protocols for many commonly used cell types. However, these conditions may have to be modified slightly for your particular cell line, since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions listed should be understood as a starting point for your own optimization. For cell lines that are not listed in our database, there is a pre-programmed 24-well optimization protocol built into the Neon device.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Do the Neon instrument settings that work well for plasmid DNA also work for siRNA?

In most cases, instrument settings that were optimized for a certain cell line or primary cell type using a plasmid will also work with siRNA. However, those settings may not be the most optimal ones for the delivery of siRNA. Therefore, additional optimization may be required to improve knockdown efficiency of the target. For cell lines or primary cell types that have not been optimized with plasmid DNA, a 24-well optimization is the best approach to find optimal conditions. Keep in mind that for every condition tested, a negative control siRNA needs to be transfected in order to normalize knockdown efficiency.

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

A good start is to use the plasmid electroporation parameters for the same cell type. 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) contains optimized plasmid electroporation parameters for many commonly used cell types. If the Neon Cell Database does not contain your cell type of interest, you can use the 24-well optimization protocol that is pre-loaded on the Neon device. Please contact Technical Support at techsupport@thermofisher.com if you should need further assistance.

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How is siRNA concentration determined in Neon transfection?

The siRNA concentration in Neon transfection refers to the siRNA concentration in the culture medium and not to the siRNA concentration in the electroporation content in the Neon tip. For example, if electroporation is performed with the 100 µL Neon tip and the transfected cells are plated in a 24-well plate that contains 500 µL culture medium, the siRNA concentration is measured as the concentration in the 500 µL culture medium and not the concentration in the 100 µL electroporation content.

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Can I use the Neon Transfection System for RNAi applications?

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 pre-programmed 24-step optimization protocol.

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How many times can the Neon Electrolytic Buffers (E and E2) be used?

If using the same plasmid/siRNA and the same cell type, one can use the Electrolytic Buffer for up to 10 times and then change the tube and buffer together. If a different plasmid/siRNA or cell type is used, we recommend changing the buffer after one usage to avoid carryover contamination.

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Can I wash and reuse Neon tips?

We strongly advise against washing the Neon tips. Washing will not remove DNA or siRNA attached to the tip and will increase the risk of cross-contaminating the samples. Also, the tips cannot be sterilized, easily increasing the risk of microbial contamination of cultures.

To avoid contamination caused by carryover from sample to sample, we recommend that you do not re-use the tip. However, if performing a 24-well optimization or transfections performed in duplicate, the tips may be used up to 2 times. The reason for this recommendation is that some of the gold coating the wire electrode inside the tip is released each time an electrical pulse is delivered. Therefore, repeated use of the tip will result in a thinning of the gold coating, causing the conductivity of the tip to change. We found that this effect becomes measurable after three uses. To ensure correct voltage and current are delivered for every electroporation, use the tip only twice.

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What is the maximum number of times I can use each Neon Transfection Tube?

The Neon Transfection Tubes are disposable and we recommend using each tube for a maximum of 10 times for the same plasmid/siRNA/cell type, to minimize the possibility of cross-contamination. In addition, we strongly recommend that a new Neon tube be used for a different plasmid DNA/siRNA or cell type, to avoid cross-contamination. If you need extra Neon tubes to accommodate your experiment, they can be purchased separately (Cat. No. MPT100).

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

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 much less than a custom service would.

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

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How can I use the Neon system for cells that are not found in your current Neon Cell Database?

We recommend reviewing our Neon Transfection System Protocols and Cell Line 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) to identify a cell type that is similar in tissue origin, to your own cell and try the recommended parameters in the protocol. This is a good starting point, but some optimization may be needed (ex. adjusting voltage). For example, if you have 293 T cells and you find a protocol for HEK293 cells in the Neon Cell Database, you can use the electroporation parameters of HEK293 cells for 293 T cells, since both are derived from human embryonic kidney.
- You can use the pre-programmed 24-well optimization protocol in the Neon device to optimize conditions for your cell type.
- Contact Technical Support at techsupport@thermofisher.com for further discussion.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why should antibiotics such as Pen/Strep be left out of the post-electroporation culture medium?

Application of a strong electrical field weakens the cell membrane and induces pore formation, which allows the antibiotics to enter the cells. Cell death is induced most likely via toxic metabolic intermediates. In addition, streptomycin has been shown to bind to eukaryotic ribosomes and may directly interfere with protein translation. If your cells need to be cultured in the presence of antibiotics, you can add them back a few hours after electroporation.

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How does plasmid size affect cell viability?

The cytotoxic effects observed with some large plasmids are often not related to their size, but are more likely due to sequence-specific effects, contaminants carried over during plasmid preparation (ex. LPS), or very large DNA amounts delivered during electroporation. Always use anion-exchange chromatography-based kits (such as our PureLink HiPure, PureLink HiPure Expi, or PureLink Expi Endotoxin-Free Plasmid Isolation Kits) to prepare transfection-grade plasmid DNA. During plasmid DNA isolation, avoid overloading the columns, as this will result in plasmid preparations of low purity.

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Is there a maximum plasmid size that can be efficiently transfected using the Neon system?

In general, electroporation is a size-dependent transfection technique and transfection efficiency declines as plasmid size increases. 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. Preliminary results indicate that bacterial artificial chromosomes (BACs) can be transfected as well, but with a low transfection efficiency. Keep in mind that in terms of molarity, 1 mg of a 6 kb plasmid corresponds to 2 mg of a 12 kb plasmid. This is why plasmid size is taken into consideration when comparing transfection efficiencies between plasmids of different lengths. For example: when comparing the transfection efficiency between 1 mg of a 10 kb plasmid and the transfection efficiency of a 150 kb BAC, 15 mg of the BAC would have to be used. This is not feasible since DNA amounts that large will cause cytotoxicity. On the other hand, this does not mean that BACs cannot be transfected using the Neon system. However, transfection efficiencies with a large amount of DNA will be very low.

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I am planning to use the Neon Transfection System. Does linearization improve stable integration of my plasmid?

Circular and linearized plasmids (that do not contain special recombination sequences) transfect into the cell and integrate into the genome with similar efficiencies. However, the area of recombination on the plasmid can be influenced by linearization, as loose ends are preferred over continuous stretches of sequence. By linearizing the plasmid, you can determine the position within the plasmid where the recombination occurs, thereby conserving the expression cassette in most cases.

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

We do not have in-house data, but several reports from customers using a variety of cell lines suggest that it works.

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What plasmid preparation method do you recommend for the Neon system?

We recommend using anion-exchange chromatography to prepare transfection-grade plasmid DNA. This technology is the basis of our PureLink HiPure line of plasmid purification kits. For large plasmids (>50 kb), do not use the PureLink HiPure purification kits that contain filters or precipitators to avoid damage to your plasmid. We do not recommend using spin columns for plasmid purification, as they contain silica membranes that do not remove impurities to the same extent as anion-exchange resins. For high-yield plasmid DNA isolation, we reccomend the PureLink HiPure Expi or the PureLink Expi Endotoxin-Free Plasmid Purification Kits.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the Neon Transfection System for delivery of siRNA into exosomes? What parameters could I try?

Currently, we do not have a validated protocol.

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Can I use the Neon Transfection System for the electroporation of cells other than mammalian cells? How about bacterial cells?

The Neon Transfection System may be used for electroporation of Chlamydomonas. Please refer to the GeneArt Chlamydomonas Protein Expression Vector (Cat. No. A24231) manual for Neon instructions. Currently, we do not offer a Neon protocol for electroporation of bacterial cells.

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Can I use the Neon electroporation technology for cell fusion applications?

Cell fusion may occur "accidentally" as a side-effect during transfection using the Neon system, for some cell-types that tend to form cell clusters (e.g., PC-12 cells), but unfortunately, we do not offer a Neon program that is optimized for cell fusion applications.

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What is your control plasmid for the Neon Transfection System and how is it purified?

Although our Neon Transfection kits do not include a control plasmid, the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146) may be used as a transient expression control.

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Do you have any recent publication or reference list where others have used the Neon instrument for their cells?

Yes. You may access the Neon citations here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-citation.html/).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I optimize my electroporations using the Neon Transfection System?

For each cell type in our 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), we offer validated electroporation parameters optimized with a universal electrolytic buffer. These conditions may have to be modified slightly for your particular cell line, since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions listed should be understood as a starting point for your own optimization. For cell lines that are not listed in our database, there is a 24-well pre-programmed 24-well optimization protocol built into the Neon device.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How should I clean the Neon device and the Neon Pipette Station?

For routine cleaning, we recommend cleaning the surface of the Neon device and Neon Pipette Station with 70% ethanol. Do not use harsh detergents or organic solvents to clean the unit. For deeper cleaning, use a broad-range medical disinfectant. Avoid spilling any liquid inside of the Neon Pipette Station. For accidental spiils (e.g., buffer, water, coffee) inside the Neon Pipette Station, disconnect the station from the main device and wipe the station using dry laboratory paper. Invert and leave the station for 24 hours at room temperature for complete drying. Do not use an oven or autoclave to dry the Neon Pipette Station.

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How should I calibrate the Neon Pipette?

The Neon Pipette is permanently calibrated by the manufacturer and does not require any further calibration.

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Can I order any of the Neon kit components separately?

The Neon transfection tubes are available separately (Cat. No. MPT100).

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What is the shelf life of the Neon kits?

The shelf life of the Neon kits is a minimum of 4 months from the date of purchase.

How long is the interval between pulses in the Neon system?

The interval between pulses is fixed at 1 ms.

What type of pulse wave does the Neon device use?

The Neon device uses a square pulse wave; the pulse width range is 1-100 ms and the pulse number range is 1-10. Volts range from 500-2500 V.

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Can I get the composition of Neon E, E2, R, and T buffers?

All buffer compositions are proprietary.

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What is the difference between the Neon E and E2 Electrolytic buffers?

The E2 buffer has higher osmolarity than E buffer. Higher osmolarity prevents the leakage of electroporation content from the 100 mL Neon tip, which has a aperture hole at the tip end than the 10 mL Neon tip (pore diameter of the Neon tips: 100 mL tip = 2.10 mm; 10 mL tip = 0.65 mm).

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I ran out of Neon Resuspension Buffer R; do you offer this as a stand-alone item?

Sorry, we do not offer Resuspension Buffer R as a stand-alone item.

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When should I use the Neon "T" buffer instead of "R" buffer?

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 high voltage (>1800 V) is applied to buffer R, sparks or arcing may be observed, regardless of cell number and other conditions. The maximum voltage for R buffer is approximately 1900 V. Buffer T composition differs from that of Buffer R to allow the application of higher voltages due to lower conductivity.

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

Both R and T Resuspension Buffers are used to resuspend cells prior to electroporation. Resuspension Buffer R is used for established adherent and suspension cells as well as primary adherent cells. Resuspension Buffer T is an alternative cell resuspension buffer for primary T and B cells, PBMCs, monocytes, and bone marrow-derived cells. Buffer T differens in composition 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.

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

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.

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How does the Neon Transfection System compare to other delivery methods (i.e., lipid-based transfection, viral delivery)?

The Neon Transfection System delivers DNA, mRNA, siRNA, and proteins efficiently into all mammalian cell types and is the best option when lipid-based transfection efficiencies are low. For immune or blood-derived cells, we generally recommend the Neon Transfection System. For neuronal cells, we recommend mRNA delivery with Lipofectamine MessengerMAX Transfection Reagent. For all stem cells, we recommend Lipofectamine Stem Transfection Reagent. Please visit our Transfection Reagent Selection Guide: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html Lentiviruses are also very efficient in the delivery of vectors for all cell types for gene expression and RNAi applications. We recommend producing high titer lentivirus particles using our LV-MAX Lentiviral Production System.

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

Unlike standard cuvette-based electroporation chambers, the Neon system uses a patented 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 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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What is the difference between the Microporator (MP-100) from Digital Bio/NanoEntek and the Neon Transfection System?

The Neon Transfection system is the second generation of the Microporator (MP-100) from Digital Bio/NanoEntek. Both instruments feature a unique pipette tip electrode chamber instead of a standard electroporation cuvette. The performance of these instruments is comparable, but the Neon Transfection System has the following improved features:

- Improved user touchscreen interface with larger display area
- Most updated firmware (available at Instrument Management: https://www.thermofisher.com/us/en/home/products-and-services/services/instrument-qualification-services/instruments-and-services-portal.html)
- Pre-programmed with one 24-well optimization protocol
- Improved sensor connector design
- >130 validated cell-specific online protocols

We do not carry an adapter to allow compatibility between former and current devices. The kits and components in both systems are exactly identical except that they are branded as Neon in the Neon Transfection System. The old manual can be used for the Neon Transfection System instrument. More information regarding the history of the Neon Transfection System can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/history-of-the-neon-transfection-system.html).

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Which special features of the Neon Transfection System are superior to those in other standard electroporation devices?

The Neon Transfection System has the following unique advantages:

1. Pipette tip chamber design for easy cell handling, uniform electric field, and higher viability due to minimal pH change during electroporation.
2. Single transfection kit (Neon Kit) compatible with all mammalian cell types, including primary and stem cells, thereby avoiding the need to determine an optimal buffer for each cell type. Only two cell resuspension buffers cover all cell types: T buffer for primary T and B cells, PBMCs, monocytes, and bone marrow–derived cells, and R buffer for established adherent and suspension cells as well as primary adherent cells.
3. Easily scalable for small or large cell culture formats. Use as few as 1 X 10E4 or as many as 5 X 10E6 cells per electroporation in a sample volume of either 10 µL or 100 µL.
4. Over 130 validated online protocols, optimized for ease of use and simplicity. Visit our 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)
5. Pre-programmed with a 24-well optimization protocol to quickly identify best electroporation parameters by cell type.
6. Flexible payload and applications. Delivers DNA, mRNA, siRNA, and proteins.

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Will nicked DNA lead to reduced transfection efficiency using the Neon device?

Yes. We recommend verifying the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid. The content of “nicked” DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in a significant decrease in transfection efficiency.

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

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

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I am using the Neon Transfection System and my R buffer has a precipitate. Will the precipitate dissolve in a 37 degrees C water bath?

No, the precipitation is irreversible. Please contact Technical Support at techsupport@thermofisher.com to obtain a replacement, if the Neon kit was purchased within 1 year.

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What are the important considerations for transfection of large plasmids using Neon electroporation?

We recommend the following for large plasmid transfection using the Neon Transfection System:

- Prepare highly concentrated plasmid (i.e., 5 mg/mL) to keep DNA volume less than 10% of the total electroporation reaction.
- Use pure plasmid to avoid cytoxicity issues. Confirm that A260/280 and A260/230 ratios are between 1.6-2.0. For endotoxin-free plasmid DNA, we recommend the PureLink Expi Plasmid Purification kit.
- Keep DNA quantity to ranges recommended in the product manual for the 10 or 100 µL tip. Some optimization is normal based on variability in cell size, cell density, and plasmid.
- Confirm DNA integrity and absence of degradation by agarose gel electrophoresis.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can you provide some tips for determining siRNA transfection efficiency with the Neon Transfection System?

To determine the Neon transfection efficiency for siRNA, we recommend transfecting the cells with a fluorescent-labeled negative control siRNA (BLOCK-iT Fluorescent Oligo, Cat. No. 13750062) and measuring the transfection efficiency as the percentage of fluorescent stained cells among viable cells. However, keep in mind that there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescence detection with a microscope does not distinguish the siRNA that enters the cell from the siRNA that may adhere to the cell membrane. Instead, we recommend transfecting with a positive control siRNA that targets a housekeeping gene such as GAPDH and measure mRNA or protein knockdown.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why do my monocytes/macrophages get activated upon transfection with your Neon system?

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. Use plasmid DNA that has been purified by anion-exchange chromatography, such as our PureLink HiPure Plasmid. PureLink Expi, or Purelink Expi Endotoxin-Free Purification Kits. If you still observe activation, you may subject your plasmid to a second round of anion-exchange chromatography purification. If you still get activation, the plasmid itself may contain sequences that 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 to be endotoxin-free and do not cause monocyte/macrophage activation in our hands.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I am experiencing arcing issues with the Neon Transfection System-what could be causing this?

Arcing could be caused by high voltage or pulse length settings, high salt or contaminants in the DNA sample, incorrect cell density, and bubbles formed during mixing of cell samples. We recommend performing the 24-well optimization (see product manual) to identify the best electroporation parameters for your cell type. Please ensure that plasmid DNA A260/280 and A260/230 ratios are between 1.6-2.0. Use either a hemacytometer or Countess II Automated Cell Counter to accurately determine cell density. Mix samples gently to avoid bubble formation and pipette samples in a slow, smooth motion to avoid air uptake.

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What is your recommended strategy for optimizing siRNA transfection using the Neon system?

For initial measurement of siRNA uptake, our FITC-labeled BLOCK-iT Fluorescent Oligo for electroporation (catalog number: 13750062) can be used. However, the fluorescent signal is somewhat weak and it is not easy to determine whether the oligo has actually entered the cells or is just sticking to the outside of the plasma membrane. In addition the signal fades quickly. Therefore, a better strategy is to use qPCR to measure knockdown of an actual target mRNA such as GAPDH and normalize it to a scrambled negative control.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I increase cell viability of Dendritic cells after electroporation?

DNA amount and quality are very critical for electroporation of Dendritic Cells (recommended 0.5-1µg; maximal 2 µg). LPS (lipopolysaccharides) have a strong negative effect on cell viability. Please make sure that all components you use for dendritic cell culture and transfection, e.g. PBS, FCS and especially DNA, are LPS free. We additionally recommend purifying your DNA by precipitating it twice with 20% PEG/2.5 M NaCl. Viability is usually not an issue when working with mRNA or siRNA.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Do you have suggestions for improving viability when transfecting HL-60 cells with the Neon system?

Our online protocol suggests to use 50,000 cells with the 10 microliter tip. However, HL-60 cells do not survive well when transfected at low density. To improve viability you can try using at least 100,000 (better: 200,000) cells with the 10 microliter tips. Also, make sure your plasmid DNA is highly purified, as HL-60 cells are sensitive to LPS. If you observe that viability is good after 24 hours but decreases over the next 72 hours, you may either be using the wrong culture medium (RPMI 1640 + 10% FBS is recommended, do not use DMEM) or your batch of FBS contains low levels of cytotoxic contaminants.

Other possibilities are mycoplasma contamination or very high passage number of your cells. If this is the case please buy a fresh vial of HL-60 cells from ATCC and try your transfections again.

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Does efficiency vary with 10 ul and 100 ul tips in the Neon System?

There is no reason to speculate that an optimized electroporation parameter would be different between 10 ul and 100 ul tips. Therefore, even though the table in the Neon cell database states 10 ul tip, those conditions should be OK for 100 ul tip also as long as the density of the cells remains the recommended one. And if the result of 100 ul is much worse than 10 ul, change voltage either increasing or decreasing by 50 to 100V. It should work well with this slight adjustment of voltage.

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

For large plasmids, it is important to prepare a highly concentrated solution of the plasmid for electroporation. An example: For the control plasmid, 0.5 ug is used for a 10 ul electroporation. The size of the control plasmid is about 5.5 kb. If your large plasmid is 50 kb, then the size is almost 10 times larger than the control and you have to use 10 times more weight of plasmid to have an equal number of molecules present. So you will need 5 ug of the large plasmid in a 10 ul electroporation to have an equivalent number of plasmids present. In order to keep the volumes low, the plasmid concentration will have to be over 5 mg/ml.

One thing to keep in mind is that when you add high amount of plasmid, it can damage cells due to toxicity of plasmid sample itself. So you will need to optimize the plasmid amount. A recommended strategy would be to start by adding the 50 kb example plasmid up to 2 ug per each 10 ul electroporation. Even though theoretically you should add more than 2 ug due to the large size of plasmid, start with 2 ug to avoid any toxicity. Then check the result. If viability looks OK and efficiency is lower than expected, increase the amount of plasmid in further reactions until you find the optimal amount for best results.

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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?

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

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

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Will nicked DNA lead to reduced transfection efficiency?

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid.

The content of "nicked" DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in significant decrease of transfection efficiency.

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What are the most common causes for low cell survival rate using the Neon device?

Here are some possibilities:
(1) Sub-optimal electrical parameters
(2) Poor plasmid quality such as endotoxin contamination
(3) Plasmid preparation containing high salt
(4) Plasmid quantity too high
(5) Cells are stressed or damaged
(6) Multiple uses of the same Neon tip

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What are the most common causes for low transfection efficiency when using the Neon device?

Here are some possibilities:
(1) Sub-optimal electrical parameters
(2) Plasmid preparation containing high salt
(3) Plasmid larger than 10 kb
(4) Plasmid concentration too low
(5) Cells are stressed, damaged or contaminated by Mycoplasma
(6) Cell density too low or too high
(7) Cells with high passage number

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Can I transfect adherent cells on a plate using the Neon transfection system?

You cannot. You need to lift the cells off of the plate through a normal method such as that used to “passage” the cells (e.g., trypsin), perform the transfection in the Neon tip, and re-plate the cells in the plate after transfection.

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

The optimal time for protein analysis is related to the stability of the protein being expressed. For a short-lived protein, like luciferase, analysis 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.

Would Neon system transfection affect functional study of membrane proteins?

As electroporation uses electric shock to make pores on cell membrane, this can damage certain membrane proteins. But those membrane proteins will generally be re-expressed and recover as time goes by. The recovery time may vary and will depend on the type of cell and protein; there is no general guideline for this yet.

An exception are primary cells which do not proliferate after electroporation, where the membrane damage could be permanent such that it hinders certain membrane protein studies. However, this is quite rare. Given sufficient time to recover, for most proliferating cells there should be no problem with membrane proteins.

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Did Invitrogen develop the Neon transfection chamber technology?

No. This technology was originally developed by a Korean company named Digital Bio Technology and marketed under the name “microporation”. Since its inception in 2006 there have been over 40 publications describing the use of this electroporation technology. Please go to www.microporation.com for further information. Invitrogen is now the only provider selling a re-designed microporator under the name Neon Transfection System.

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What is the minimum time I should wait before adding antibiotics to the culture after electroporation?

After electroporation, wait for about 4-6 hours before adding antibiotics back to the cells. This is to make sure that the membrane integrity has been restored.

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

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.

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When do you use Neon Buffer T?

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.

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Do Neon instrument settings which work well for plasmid DNA also work for siRNA?

In most cases instrument settings for a certain cell line or primary cell type which were optimized using a plasmid will also work with siRNA. However, those settings may not be the best possible settings for the delivery of siRNA. Therefore, additional optimization may be required to improve knockdown efficiency of the target. For cell lines or primary cell types which have not been optimized with plasmid DNA, a 24-well optimization is the best approach to find efficient conditions. Keep in mind that for every condition tested, a negative control siRNA needs to be transfected in order to normalize knockdown efficiency.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can the Neon instrument settings determined for the 10 microliter tips be used with the 100 microliter tips?

In most cases they can, but in some cases reduced transfection efficiency may be observed and adjustment of the voltage settings may be required to improve efficiency. A 24-well optimization with the 100 microliter tips is usually not necessary.

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What is the maximum number of cells one can transfect in a 100 ul Neon System tip?

According to Neon guidelines, you can run up to 8 million cells in 100 µL. If the cells are small enough, up to 10 million cells can be used in 100 µL.

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

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.

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

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.

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Can the Neon System be used for co-transfecting two or more plasmids?

We currently do not have data to support this application, but co-transfection of different plasmids is possible. 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 total DNA followed by a stepwise increase. Various ratios of the plasmids should be tested if toxicity is observed

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Do you need to change the Neon electrolytic buffer tubes when changing cells or constructs?

The electrolytic buffer tubes are disposables and you should change 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 plasmid 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. In case you run out, the tubes can be purchased separately (Catalog number: MPT100).

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Where can I find Neon instrument settings for my cell line?

Go to our "Learn More about the Neon Transfection System" page at www.thermofisher.com/neon. There you will find a link to our cell transfection database which currently contains in-house optimized settings for over 140 cell lines and primary cells. However, these conditions may have to be modified slightly for your particular cell line since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions which we have posted on our website should be understood as a starting point for your own optimization.

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How many times can a Neon Tip be used?

To avoid contamination caused by carry-over from one sample to another, we recommend that you do not use the Neon Tip for more than 2 times. Oxide formation at the piston surface area can be generated if the tips are used more than 2 times, which decreases electrode function of the piston.

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How often can you use the Neon tips?

We recommend using the tips 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 conductivity of the tip to change. If you want to be absolutely sure that the correct voltage and current are delivered to your cells, use the tip only twice.

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How do you determine transfection efficiency and cell viability?

We typically stain suspension cells with 0.5% propidium iodide, then gate viable and non-viable populations using a Guava 96-well PCA (Millipore). Transfection efficiency obtained with our EmGFP-expressing control plasmid is calculated based on the total population of cells. This is in contrast to Amaxa which calculates efficiency with respect to the viable cell population. For adherent cells, we trypsinize and then pool trypsinized and non-adherent cells from the supernatant prior to staining with propidium iodide.

If you do not have a Guava PCA system or FACS, you can determine viability and transfection efficiency using the trypan blue exclusion method and a fluorescence microscope with filter settings appropriate for the detection of GFP (emission: 509 nm). However, this approach is less accurate than FACS and much more tedious.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How does plasmid size affect cell viability in transfection?

There is no evidence that larger plasmids cause increased toxicity in all cases. The toxic effects that are seen with some large plasmids are not related to their size, but are likely due to sequences located on the plasmid or to contaminants in the plasmid preparation, such as LPS. Always use anion exchange chromatography-based kits (such as Invitrogen’s PureLink HiPure kits) to prepare transfection grade plasmid DNA and avoid overloading the columns, as this will result in plasmid preparations of low purity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the optimal size of DNA that you recommend for electroporation with the Neon transfection system?

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

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What is the best time for analysis of siRNA knockdown?

As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA half-life can range from minutes to days (Ross J, 1995, Microbiol Rev 59:423-50) while the half-life of protein products can range from less than a few minutes to several days. In general, the recommended time course ranges from 12 to 72 hours to deplete target mRNA and 24 to 96 hours to adequately knock down target proteins.

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

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How is Neon System siRNA transfection efficiency determined?

To determine the Neon System transfection efficiency for siRNA, the cells are transfected with a fluorescent-labeled negative control siRNA; the transfection efficiency is measured by the percentage of fluorescent stained cells among all cells, both live and dead. We recommend to use the BLOCK-iT Fluorescent Oligo for electroporation from Invitrogen (Cat. No. . 13750062). However, there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescent detection with a microscope does not distinguish the siRNA that goes into the cell from the siRNA that sticks to the cell membrane. To obtain a more accurate transfection efficiency, one needs to transfect the cells with a positive control siRNA such as the one that targets a house-keeping gene and measure the knockdown of target RNA or protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why do you recommend that the Neon Tube is used for a maximum of 10 times?

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

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

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

(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

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

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

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Does linearization improve stable integration of my plasmid?

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.

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

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

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

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.

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

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.

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Can I use the Neon electroporation device for RNAi applications?

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.

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Can I use electroporation technology for cell fusion applications?

Cell fusion may occur "accidentally" as a side-effect during transfection for some cell-types which tend to form cell clusters (e.g. PC-12 cells), but unfortunately, we do not offer a Neon system program to optimize for cell fusion.

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How does the Neon system compare to other delivery methods (ex. Lipids, Viral Delivery)?

The Neon Transfection System efficiently delivers nucleic acids (DNA/siRNA) into most mammalian cell types, including traditionally difficult-to-transfect cells like primary cells and neurons. In comparison, lipid transfection reagents are much less efficient for such cell lines, and in some cases do not work at all. While viral delivery is also generally effective for difficult-to-transfect cells compared to lipids, it can be much more labor intensive and time consuming than electroporation with the Neon Transfection System, especially if you have many different DNA or RNAi molecules to transfect and screen.

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How is Neon System transfection efficiency measured?

We normally analyze transfection efficiency in living cells by FACS. We first exclude cellular debris by gating for the "normal" population (regarding size and granularity) in the forward-sidescatter. From this gated population we determine dying cells by propidium iodide staining and exclude them from analysis by setting another gate. For example, in one experiment we transfected Human primary CD4+T-cells with 2250V, 20ms, 1 and 0.5 ug EGFP in primary blood buffer using the Neon System. 24 hrs post electroporation, cells were analyzed by flow cytometry. CD4+T-cell were gated according to forward/side scatter, and dead cells were excluded by propidium iodide staining and gating. GFP gene expression of T cells was measured after electroporation with plasmid DNA, and we found 32.74% transfection efficiency and 75% viability. Please see our "Learn More about Neon Transfection System" pages (search this term from our home page) for more data and information.

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How are the Neon Kit cell viability and transfection efficiency determined by your R&D team?

Cells were analyzed for viability and transfection efficiencies 24 hours or 48 hours post-electroporation using a Guava PCA-96 Cell Analysis System. LIVE/DEAD cell viability assay populations were calculated by propidium iodide staining (1:2000). The percent of transfected cells was calculated by dividing the number of GFP positive cells by the total population and recorded as transfection efficiencies. The percent of dead cells was calculated by dividing the number of PI-stained cells by the total population

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Is there a difference in how transfection efficiency is measured with the Neon System versus the Nucleofector Device?

The Neon system measures the transfection efficiency by the percentage of GFP-positive cells among all cells which include live and dead cells. In contrast, the Nucleofector Device measures the transfection efficiency by the percentage of GFP-positive cells among only the live cells.

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How should I clean the Neon device and the Pipette station?

Clean the surface of the Neon device and Neon Pipette Station with a damp cloth. Do not use harsh detergents or organic solvents to clean the unit. Avoid spilling any liquid inside of the Neon Pipette Station. If you accidentally spill any liquid (e.g., buffer, water, coffee) inside the Neon Pipette Station, disconnect the station from the main device and wipe the station using dry laboratory paper. Invert and leave the station for 24 hours at room temperature for complete drying. Do not use an oven to dry the Neon Pipette Station

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What are the benefits of the Neon Transfection System over standard electroporation?

(1) Small to large number of cells can be used. The transfection is performed using as few as 2 x 10E4 or as many as 8 x 10E6 cells per reaction using a sample volume of 10 ul or 100 ul.
(2) The Neon Transfection System uses a single transfection kit (Neon 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. Two cell suspension buffers cover all cell types: T buffer (not yet included in the kit) for primary T and B cells, and R buffer for other cells.
(3) Open and transparent protocols that are optimized for ease of use and simplicity. The Neon Cell Database (www.thermofisher.com/neon) contains optimized protocols for many commonly-used cell types.
(4) The Neon device is preprogrammed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type.

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Does Invitrogen have an optimization service for the Neon System?

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.

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What is your Neon control plasmid and how is it purified?

Our control plasmid is a 5.9 kb pcDNA6.2-based expression construct expressing EmGFP from a CMV promoter. The purification procedure is proprietary, but the purity of this plasmid is equivalent to two rounds of anion exchange chromatography.

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Can you wash and re-use the Neon tips?

We do strongly advise against washing the tips. Washing will not remove DNA or siRNA attached to the tip and will increase the risk of cross-contaminating your samples. The tips can not be sterilized easily after cleaning, thus increasing the risk of microbial contamination of your cultures.

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Do Neon System Tips fit with NanoEntech’s pipette?

Yes, they do.

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What is the composition of the E, E2, R and T buffers for the Neon System?

All buffer compositions are proprietary.

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What is the difference between E (Electrolytic) buffer and E2 buffer for the Neon Transfection System?

The E2 buffer has higher osmolarity than E buffer. Higher osmolarity prevents the leakage of electroporation content from the 100 ul Neon tip which has a larger hole at the tip end than the 10 ul Neon tip (pore diameter of the Neon tips: 100 ul tip = 2.10 mm; 10 ul tip = 0.65 mm).

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What is the difference between the “R” buffer and the “E" or "E2” buffers for Neon?

The “R” buffer is used to resuspend the cells prior to transfection (“R” for Resuspension”). The “E or E2” buffers are the electrolytic buffers used for electroporation. “E or E2” buffers are added to the Neon tube prior to electroporation

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What is the shelf life for the Neon Transfection System 100 μL Kit (Cat. No. MPK10096, MPK10025)?

There is no expiration date for the Neon Transfection System 100 μL Kit (Cat. No. MPK10096, MPK10025). However, Thermo Fisher Scientific does warranty the product for 12 months after it has been shipped to the customer.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are the tips from the Neon Transfection System 100 μL Kit (Cat. No. MPK10096, MPK10025) available separately?

No, the tips within the Neon Transfection System 100 μL Kit (Cat. No. MPK10096, MPK10025) are not available as a stand-alone product.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Do you have a Chlamydomonas transformation protocol using the Neon Transfection System?

We recommend the following guidelines:

- For high transformation efficiencies, the OD750 of the C. reinhardtii culture should be > 0.8 before electroporation.
- Carry out all transformation steps at 4 degrees C using solutions pre-equilibrated at 4 degrees C.
- Electroporate the cells using the 100 µL Neon tip in TAP-40 mM sucrose solution or the MAX Efficiency Transformation Reagent For Algae (Cat. no. A24229) at 4 degrees C.
- Use the following Neon electroporation parameters: 2300 volts (Voltage), 13 ms (Pulse Width), 3 (Pulse Number)

For detailed instructions on using the Neon Transfection System, refer to the Neon Transfection System User Guide (https://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.