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

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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.

Answer Id: E9007

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What are the special features of the Neon™ Transfection System that make it superior to standard electroporation?

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Here are the special features of the Neon™ transfection System: 1. 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 resuspension buffers are provided to 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.
2. Small to large numbers of cells can be used in the Neon™ Transfection System. The electroporation is performed using as few as 1 × 10E4 or as many as 5 × 10E6 cells per reaction using a sample volume of 10 μL or 100 μL.
3. Open and transparent protocols are available, that are optimized for ease of use and simplicity. 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 protocols for many commonly used cell types.
4.The Neon™ device is pre-programmed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type.

Answer Id: E9077

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What is the difference between the Microporator (MP-100) from Digital Bio/NanoEntek and the Neon™ Transfection System?

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The Neon™ Transfection system is the second generation of the Microporator (MP-100) from Digital Bio/NanoEntek. The unique feature of both these instruments is that they use an electronic pipette tip as a transfection chamber instead of a standard electroporation cuvette. The performance of these instruments is comparable, but there are some differences. The main difference is in the user interface. The Neon™ Transfection System unit has updated firmware and can upload more programs than the Digital Bio Microporator MP-100. The MP-100 Microporator is white in color and has a smaller screen than the Neon™ Transfection System unit. The Neon™ Transfection Pipette Station is not compatible with the MP-100 pipette (white in color). This is because the sensor connector (it is a 2-pin connection) is different from that in the Neon™ Pipette Station (12-pin connection). We do not carry an adapter to allow compatibility. 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).

Answer Id: E9078

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What is the shelf life of the Neon™ kits?

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The shelf life of the Neon™ kits is 1 year from the date of purchase.

Answer Id: E9091

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

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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 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 one that targets a housekeeping gene, and measure the knockdown of target RNA or protein.

Answer Id: E8995

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How does the Neon™ system compare with the Amaxa Nucleofector™ II?

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Learn more about the Neon™ Transfection System versus the Amaxa Nucleofector™ II System here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-vs--amaxa.html).

Answer Id: E9079

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

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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. Multiple uses of the same Neon™ tip
7. Microbubbles in tip, causing arcing

Answer Id: E9130

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Can I order any of the Neon™ kit components separately?

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No. The Neon™ kits are only available as full kits for up to 50 or 192 reactions. The Neon™ transfection tubes are available separately (Cat. No. MPT100). The Neon™ kits contain sufficient buffers to perform 2 electroporations per tip.

Answer Id: E9092

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

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

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

Answer Id: E5496

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Can I use the Neon™ Transfection System for siRNA-loaded exosomes? What parameters could I try?

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We currently do not have a validated protocol. However, we could suggest some parameters to try based on a recent publication (Alvarez-Erviti et al., Nature Biotech 29(4):341-347, 2011), where cells were electroporated with a non-identified cuvette-based electroporator. The parameters used were 400 V and 127 mF. For the Neon™ system, this translates to about 1350 V. We would recommend running the optimization protocol with this voltage in mind; however, it is difficult to recommend pulse width or number of pulses—thus, optimization is necessary.

Answer Id: E9100

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

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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.

Answer Id: E9116

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

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

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

Answer Id: E5529

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

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Answer

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.

Answer Id: E5546

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

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Our control plasmid is a 5.9 kb pCDNA™6.2-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. This plasmid is not meant to be propagated, and we cannot confirm any selectable markers.

Answer Id: E9097

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

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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.

Answer Id: E5523

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