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MPK1096

Product FAQ

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

Answer

Yes. To check the quality of your DNA, we strongly recommend determining the A260:A280 ratio. It should be at least 1.6 for a good DNA preparation.

Answer Id: E5538

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Product FAQ

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

Answer

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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Product FAQ

Can I use electroporation technology for cell fusion applications?

Answer

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.

Answer Id: E5494

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Product FAQ

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

Answer

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

Answer Id: E5539

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Product FAQ

Can I use the Neon™ electroporation device for RNAi applications?

Answer

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.

Answer Id: E5495

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Product FAQ

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

Answer

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.

Answer Id: E5540

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Product FAQ

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

Answer

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.

Answer Id: E9098

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Product FAQ

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

Answer

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.

Answer Id: E5490

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Product FAQ

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

Answer

We recommend cleaning 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. In case 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.

Answer Id: E9094

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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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.

Answer Id: E5522

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Product FAQ

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

Answer

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

Answer Id: E9108

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Product FAQ

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

Answer

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.

Answer Id: E5541

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Product FAQ

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

Answer

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.

Answer Id: E9122

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Product FAQ

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

Answer

Currently, we do not offer a Neon™ protocol for electroporation of bacterial cells. However, the Neon™ Transfection System may be used for electroporation of Chlamydomonas. Please refer to page 18 in the GeneArt™ Chlamydomonas Protein Expression Kit manual (https://tools.thermofisher.com/content/sfs/manuals/geneart_chlamydomonas_protein_expression_man.pdf) or page 15 in the GeneArt™ Chlamydomonas Engineering Kit manual (http://tools.thermofisher.com/content/sfs/manuals/geneart_chlamy_kits_man.pdf) or page 20 in the GeneArt™ Chlamydomonas TOPO™ Engineering Kit manual (http://tools.thermofisher.com/content/sfs/manuals/geneart_chlamy_topo_kits_man.pdf) for electroporation.

Answer Id: E9099

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