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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.

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

Antibiotics should be added 24-48 h after Nucleofection.

How often should I change the medium during selection of stable clones after Nucleofection™?

Every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.

How many stable clones will I get from one transfection?

This depends on the respective cell type (transfection efficiency, viability and integration frequency). If it is important to you to know the frequency of stable integrants before carrying out your actual experiments, the frequency should be...

Following Nucleofection™, I cannot obtain stable clones from single cells, what could be the reason?

Some cell lines need certain cell densities to proliferate. For those cells you can try using conditioned medium for setting up limiting dilution or culturing single cells together with feeder cells.

What is the function of retinoic acid in the BEGM™ media? Under what conditions would you use culture NHBE cells without retinoic acid?

In vivo, bronchial epithelial cells differentiate along an abnormal squamous pathway under conditions of retinoid deficiency. The squamous phenotype is characterized by the induction of specific markers such as keratin 13, and the enzymes...

What antibiotic concentration should I use for selecting stable transfectants?

Please click on the associated file.

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...

What are mycoplasmas?

Mycoplasmas belong to the family Mollicutes, which includes Acholeplasma, Ureaplasma and other species. However the term "Mycoplasma" is most often used as a "cover-all". More than 180 species have been identified of which 20 distinct Mycoplasma and...

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