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In general, there is not a problem using IRES plasmids with Nucleofection®, with one important caveat. The levels of expressed protein for the first and second genes will not be identical, and this can create problems with analysis and interpretation...
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The original attenuation of the IRES sequence was performed to allow for a greater difference between the expression levels of the upstream gene of interest and the downstream reporter gene. If the downstream reporter gene, the product of less than...
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In general, successful Nucleofection® is vector independent, with one important caveat. Some expression plasmids utilize promoters and enhancers obtained from the Long Terminal Repeats (LTR's) of retroviruses, and when these expression plasmids are...
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GFP expression has been observed for up to one week as measured by light and fluorescence microscopy.
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As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA...
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Yes. As the same protocol applies for any nucleic acid substrate (vectors or oligonucleotides) you can easily perform co-transfections either for transfection control or rescue experiments.
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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.
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We recommend using cuvettes with the 0.1 cm gap width.
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Yes, DsRed is much less bright than GFP and is more difficult to detect by FACS and microscopy because it bleaches quickly.
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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...
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