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Regulatory statements vary by product. Please see product-specific literature to determine product use.

Can I use the Neon electroporation device for RNAi applications? Product FAQ

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.

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

Answer Id: E5495

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What protocol is recommended for delivery of Cas9 protein:guide RNA complex to my PSCs cultured in StemFlex Medium? Product FAQ

Answer

We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/PG1584-PJT1313-COL31227-Demonstrated-Protocol-Performing-CRISPR-Cas9-FHR.pdf); see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.

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

Answer Id: E14762

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

Answer

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.

Answer Id: E9001

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

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.

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

Answer Id: E9098

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

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 59:423–450) 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).

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

Answer Id: E9126

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What is the expected time constant during algae transformation? Product FAQ

Answer

The average expected time constant is 15-20 milliseconds. We have a Neon electroporation protocol in the manual.

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

Answer Id: E9565

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

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 and to allow adherent cells to attach to the culture vessel.

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

Answer Id: E9122

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

Answer

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.

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

Answer Id: E9125

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Can Lipofectamine LTX be used in transfection of Suspension cells and other hard to transfect cells? Product FAQ

Answer

We have tested transfection of suspension cells such as Jurkat and K562. We achieved transfection efficiency of less than 5% for each of them. We do not notice toxicity with Lipofectamine LTX reagent when used with these suspension cells. For other cell lines please follow the guidelines in the product insert. However, if you experience low transfection efficiency, then we recommend Neon electroporation system or Viral delivery.

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

Answer Id: E4995

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Can Lipofectamine LTX be used for transfecting primary cells? Product FAQ

Answer

We have tested four primary cell lines: HuVec, NHFF (Normal human foreskin fibroblast), MJ90 and NDHF. We have observed transfection efficiency in the range of 5-25%. We suggest optimizing transfection efficiency for your primary cell line by varying DNA and lipid ratio. If you are using any other primary cell line, please follow guidelines suggested in the product insert. Alternatively, Neon electroporation system and viral delivery methods are recommended.

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

Answer Id: E4996

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

Answer

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.

Answer Id: E13093

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Which lipid transfection reagent do you recommend for my cell line? Product FAQ

Answer

The best transfecting agent and efficiency would depend on the particular cell line you have. Please visit our online selection tools to see our recommendation for your specific cell line. If your cell line is not on the list, we recommend you try Lipofectamine LTX or Lipofectamine 2000 for plasmid transfection, and Lipofectamine RNAiMAX for siRNA transfection. For primary cells, Lipofectamine LTX with PLUS reagent is generally the best choice for plasmid transfection. For some hard-to-transfect cells, like suspension cells and stem cells, the Neon electroporation system usually works better compared to lipid transfection reagent.

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

Answer Id: E4502

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

Answer

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.

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

Answer Id: E9099

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

Answer

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.

Answer Id: E5508

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How is siRNA concentration determined in Neon transfection? Product FAQ

Answer

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.

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

Answer Id: E9114

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