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How long have you been able to observe GFP expression in primary Human Keratinocytes?

GFP expression has been observed for up to one week as measured by light and fluorescence microscopy.

Which reporter gene should I choose for my reporter gene experiment?

In general, all reporter genes can be used. For some applications (e.g. reporter gene experiments in various primary cells, experiments with late analysis time point due to starving and /or stimulation) a reporter gene with a longer half life than...

What is the maximum amount of DNA I can use in my Nucleofection Experiment?

The overall amount of 5 µg should not be exceeded. Look in the cell specific Optimized Protocol (OP) for the recommended total DNA amount of the cell type of interest.

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.

When should I begin looking for the Luciferase signal after Nucleofection?

The time course of plasmid expression after Nucleofection™ might be different from the time course seen with other transfection methods. We recommend looking at different time points and start with the first time point as early as 4 to 6 hours post...

What should I pay attention to when transferring my reporter gene set up from a cell line to a primary cell?

In general the overall expression level differs between different cell types. Don't expect the same level of gene induction when working with primary cells. You might need to change to other DNA amounts or ratios.

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 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...

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...

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...