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MPK10025

Product FAQ

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

Answer

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

Answer Id: E9129

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Product FAQ

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

Answer

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.

Answer Id: E5509

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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

What is the best time for analysis of siRNA knockdown?

Answer

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.

Answer Id: E5510

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Product FAQ

How should I calibrate the Neon™ Pipette?

Answer

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

Answer Id: E9093

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Product FAQ

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

Answer

- If you find another cell type in 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) 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. This does not guarantee that you will get the best results, but it is a good starting point. 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.

Answer Id: E9107

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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

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

Answer

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.

Answer Id: E5504

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Product FAQ

Will nicked DNA lead to reduced transfection efficiency?

Answer

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.

Answer Id: E5537

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Product FAQ

What is the minimum time I should wait before adding antibiotics 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: E5530

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Product FAQ

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

Answer

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.

Answer Id: E5511

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Product FAQ

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

Answer

For large plasmids, it is important to prepare highly concentrated plasmid. For the control plasmid, 0.5 mg is used for 10 mL electroporation and the size of the control plasmid is ~5.5 kb. Let’s say the large plasmid being used is 50 kb. This is almost 10 times larger than the control, so you would have to use 10 times more plasmid to compensate for the molecular number. So you would use 5 mg in 10 mL electroporation. To cover this large amount of plasmid, the plasmid concentration should be over 5 mg/mL. One thing to keep in mind is that when you add large amounts of plasmid, it can damage cells due to toxicity of the plasmid sample itself. Thus, we recommend optimizing, starting with a plasmid amount that is less than the amount you came up with, and then moving up in plasmid amount while checking both viability and transfection efficiency.

Answer Id: E9001

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Product FAQ

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

Answer

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

Answer Id: E5498

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