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If I use Miltenyi paramagnetic beads for positive selection of cells, i.e. beads remain on the cell surface, does that influence Nucleofection® results?

No, it seems that the beads bound to the cell surface do not have any influence on Nucleofection®. Larger magnetic beads (i.e. > 1µm) may disturb the Nucleofection® process and negatively influence cell viability and efficiency.

Which reporter gene should I choose for my reporter gene experiment?

In general, all reporter genes can be used.For some applications (e.g. reporter gene experiments in various primary cells, experiments with late analysis time point due to starving and /or stimulation) a reporter gene with a longer half life than...

Why do plasmids which contain IRES sequences often have lower transfection efficiency?

In general, there is not a problem using IRES plasmids with Nucleofection®, with one important caveat. The levels of expressed protein for the first and second genes will not be identical, and this can create problems with analysis and interpretation...

What is the amount of DNA I can use in my Nucleofection® Experiment?

This totally depends on the Nucleofector® system you are working with. While in the 100 µL reaction vessels usually 1 - 5 µg DNA is being used, for the 20 µL reaction vessels (e.g. X Unit, 96-well Unit and HT...

Why do plasmids that contain LTR sequences often have lower transfection efficiency?

In general, successful Nucleofection® is vector independent, with one important caveat. Some expression plasmids utilize promoters and enhancers obtained from the Long Terminal Repeats (LTR's) of retroviruses, and when these expression plasmids are...

Do you have a protocol for the Nucleofection® of Plasmodium berghei using the Nucleofector® Systems

Nucleofector I/II/2bWe have excellent data for the Nucleofection™ of Plasmodium berghei from the group Chris J. Janse and Andrew P. Waters from the University of Leiden. Their protocol describes a transfection efficiency of 10-3 – 10-4. With this...

When should I begin looking for the Luciferase signal after Nucleofection®?

The time course of plasmid expression after Nucleofection® might be different from the time course seen with other transfection methods. We recommend looking at different time points and start with the first time point as early as 4 to 6 hours post...

What should I pay attention to when transferring my reporter gene set up from a cell line to a primary cell?

In general the overall expression level differs between different cell types. Don't expect the same level of gene induction when working with primary cells. You might need to change to other DNA amounts or ratios.

Is it more difficult to detect DsRed by FACS than GFP?

Yes, DsRed is much less bright than GFP and is more difficult to detect by FACS and microscopy because it bleaches quickly.

Is there a difference between, for example, the programs C-20 and C-020?

No, the programs C-20 and C-020 are identical regarding their electrical parameters. The letter minus a two-digit number is the nomenclature of the Nucleofector™ I. Since we have launched the Nucleofector™ II we changed it to a letter minus a three...
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