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View additional product information for Neon™ Transfection System Pipette - FAQs (MPP100)
61 product FAQs found
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.
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.
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.
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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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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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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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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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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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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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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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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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Yes, you may co-transfect both plasmid and siRNA together at the same time but some optimization may be necessary.
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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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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.
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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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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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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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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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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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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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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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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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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.
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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.
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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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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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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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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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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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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.
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Currently, we do not have a validated protocol.
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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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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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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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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/).
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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.
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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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The Neon Pipette is permanently calibrated by the manufacturer and does not require any further calibration.
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The Neon transfection tubes are available separately (Cat. No. MPT100).
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The shelf life of the Neon kits is a minimum of 4 months from the date of purchase.
The interval between pulses is fixed at 1 ms.
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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All buffer compositions are proprietary.
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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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Sorry, we do not offer Resuspension Buffer R as a stand-alone item.
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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 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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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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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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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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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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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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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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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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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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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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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.
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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.
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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.
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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 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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The Neon Pipette is permanently calibrated by the manufacturer and does not require any further calibration.
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The metal piece that comes with the Neon Pipette is the barrel. Please watch the following video to see how it fits in the pipette and how to replace it if needed: https://www.youtube.com/watch?v=FO6xd4r__l4
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