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What are the requirements for direct Nucleofection of mRNA for protein expression?

The mRNA should be capped and polyadenylated. The conditions of Nucleofection™ will be the same as for DNA with the particular cell type, i.e. follow the same protocol and use the same program, except one will likely need to add a much higher amount...

Four hours after Nucleofection, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection™ but by 24 hours post Nucleofection™, morphology should be normal. Remember to perform a fluid change about 4 hours post Nucleofection™.

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.

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.

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.


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.

Do you have a protocol for the Nucleofection of Plasmodium berghei?

Yes. You can use our new Basic Parasite Nucleofector™ Starter kit for parasitic protozoa. The Basic Parasite Nucleofector Starter Kit (Cat.No. VMI-1001) should help you to determine the optimal program and Nucleofector™ Solution for your...
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