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Can I use the Nucleofector 2b Solutions and Optimized Protocols with the 4D-Nucleofector X-Unit or the 96-well Shuttle Device?

No. The 4D-Nucleofector and 96-well Shuttle™ System uses specifically optimized electrical parameters and solutions. Five different 4D-Nucleofector or 96-well Shuttle™ Solutions are available for primary cells. For cell lines, three different 96-well...

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.

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...

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.

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.

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...

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...

Does your Nucleofector Solutions contain anything that would inhibit attachment of adherent cells?

No. In many cases where decreased attachment is a problem, the cause is inactivated trypsin. Unless the trypsin is inactivated with trypsin inhibitor or media containg BSA or serum, it will continue to degrade the cells and ultimately decrease cell...

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.