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Generation of a stable cell line: A positive control plasmid works, but I cannot obtain stable clones with my construct of interest. What could be the reason?

One reason could be that your gene product is toxic to the cells. We recommend testing the construct for expression of the gene of interest by transient Nucleofection® of your cells. If the proportion of expressing cells drops between 4 and 48 hours...

Are there any contaminating glial cells in your Clonetics rat or mouse embryonic neuronal cells?

There will be a small amount of glial cells in the culture, these are not considered a contaminating cell type. The glial cells are necessary to help the neurons form their axons and dendrites, synchronize the activity of the axons, and remove...

Can nicked DNA lead to reduced transfection efficiency in Nucleofection® experiments?

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Compare undigested plasmid to plasmid DNA digested with a suitable single cut restriction enzyme to linearize. Supercoiled plasmid will run faster than...

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.

In stable cell line generation, 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 the generation of stable cell lines?

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.

Do your Clonetics Rat and Mouse Astrocytes proliferate?

Yes. The cells do expand in culture.

At which time point after Nucleofection® should I start selection of stable integration?

Antibiotics for selection should be added 24-48 h after Nucleofection®.

What protocol should I use for Nucleofection® of patient derived blood samples, e.g. leukemia or lymphoma cells?

Unfortunately, we do not have a ready-to-use protocol for Chronic lymphocytic leukemia (CLL) or other blood cell derived cancer cells. The cells often have a chromosomal aberration and show the properties of a cell line. Therefore we suggest to...

My suspension-adapted HEK293 (or CHO) cells tend to clump in culture. How can I avoid this?

Efficient protein production experiments require growth of the host cell in single-cell suspension, something that is sometimes difficult to achieve since most established cell lines retain their adherent growth characteristics. Commercially...
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