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How should I purify my DNA for Nucleofection of neurons?

The quality and the concentration of DNA used for Nucleofection™ in general plays a central role for the efficiency of gene transfer. We strongly recommend using endotoxin free prepared DNA. Endotoxin free Kits are available from several suppliers....

I've noticed that duration of the Nucleofector Program seems to change even when I haven't changed the program I am using. Why?

The duration time of a program is dependent on the number of the program. The duration is also dependent on how long the Nucleofector™ Device has been turned on as well as the number of Nucleofections you have done in the last couple of minutes. When...

Do I need a special incubator for cultivation of insect cells ?

Insect cells are cultivated at lower temperatures than E.coli or mammalian cells (S2 cells at 24°C, Sf9 cells at 28°C). Therefore they need a dedicated incubator with appropriate temperature.

Which promoter can be recommended for transient protein production in mammalian cells ?

Vectors with a CMV promoter [e.g. pmaxCloning™, Lonza, Cat. No. VDC-1040] typically work fine for transient protein expression in mammalian cells, like HEK293, CHO or NSO. For insect cells, like Sf9 or Sf21, vectors with insect promoters are...

What is the lowest amount of DNA which can be used for transfection? Do I need a DNA carrier for low DNA amounts?

The recommended amount of DNA per 100 µl reaction is 0.5µg-5 µg. No carrier is needed.

What is the best incubation time for transient protein production?

The optimal incubation time ranges from 2-days to 6 days depending on various factors like the stability of the protein in the medium, potential toxicity of the protein, cell density and stability of the plasmid in the nucleus. To check the timepoint...

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...

Why is the Nucleofector Technology ideal for transient protein production ?

For many cell lines in suspension, especially sCHO, conventional transfection methods are very limited in efficiency. For protein production, suspension cells are favored over adherent cells because they are much easier to cultivate. Unlike...

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.

Why is the Nucleofector Technology ideal for protein production from stable clones ?

The generation of a stable clone often requires six months or more due to selection procedures and adaption to serum-free conditions after transfection. With the help of the Nucleofector technology suspension cells can be transfected directly in a...
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