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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.

Do I need a pure neuronal culture for Nucleofection® ?

In principle, it should be absolutely fine to nucleofect pure neuron cultures (e.g. after purification by Ficoll). Glial cells are not necessary for transfection. What one would have to consider for example, is to get the required number of neurons,...

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...

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.

What are your recommendations for minimum and maximum cell numbers for Nucleofection ?

The recommended cell number will vary depending on which Optimized protocol is being used. In general, using less than 2x10exp5cells per reaction causes a major increase in cell mortality. For some cell lines we have tried cell numbers up to...

Which promoter can be recommended for transient gene expression in mammalian cells ?

Vectors with a CMV promoter 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 required. In HEK293E (EBNA) cells, the use of...

After Nucleofection®, how do you determine cell viability?

We determine cell viability after Nucleofection® in two ways: 1) FACS determination of viable/dead cells by PROPIDIUM IODIDE STAINING. We normally analyze transfection efficiency in living cells by FACS: We first exclude cellular debris by...

What is the best incubation time for transient gene expression in Nucleofection® Experiments?

The optimal incubation time ranges from 2 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 for...

Are your Nucleofector® Solutions checked for RNase activity during your QC process?

Yes. We do test the Solutions with a real time RT-PCR based assay. This assay basically measures the quantity of an RNA template after incubation in our solution. In case there is RNAse contamination the template would be digested which would be...

What is the optimal size of DNA that you recommend for Nucleofection®?

We routinely use plasmids of 4-7 kb in our laboratories and plasmids up to approximately 20kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Some preliminary results we have also...
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