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Can I use larger cuvettes for my Nucleofection Reaction ? Can I use the cuvettes more than once?

No. The electrical parameters provided by the Nucleofector™ Device are optimized for the cuvettes contained in the Nucleofector™ Kits. The cuvettes are single-use only. Using the cuvettes more than once will result in higher cell death and lower...

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.

Do you have any siRNA Nucleofection results using concentrations lower than 50nM?

Yes. Please click on the attached Technical Reference Guide: "Designing an RNAi Experiment Using Nucleofection™". On page 3 you can find a table with some examples.

How long does it take to obtain stable transfectants?

Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition.

The animal facility which isolates my hepatocytes is at a different location, should I be concerned about transport time?

Isolated hepatocytes can be transported in Krebs-Henseleit Buffer or supplemented William's E Medium. Long transport time for isolated hepatocytes, (in excess of one hour) can cause a decrease in efficiency of Nucleofection™, but does not seem to...

I have a very high transfection efficiency but most of my cells die during selection, is this to be expected?

This is the normal pattern you should expect to see. Only a small proportion (1/10,000 to 1/100) of all transfected cells will integrate the transfected DNA into their genome and become stable transfectants. The remaining cells lose the transfected...

What selection markers can I use for stable transfections?

The most commonly used marker is the neomycin phosphotransferase (neo) gene that confers resistance to G418 to eukaryotic cells. Other markers are Puromycin, Hygromycin, Zeocin, or the HPRT gene that can be used in HPRT-deficient cells.

How much DNA can I use when transfecting hepatocytes with the standard Nucleofector?

We have used up to 6 µg of DNA per 100 µl reaction with no deleterious effect.