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515 results sorted by
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The reason is that insect cells are often incubated in very simple incubators that are not humidified (and without CO2 at 24°C to 28°C). Insect media typically do not need CO2 for buffering pH, so gas exchange is not required. To avoid evaporation of...
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The ratio is 1:4,5 (500 µL of supplement is added to 2.25 mL of Nucleofector® Solution).For a single 100 µL reaction, use 18 µL of supplement plus 82 µL of solution to make the 100 µL.
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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...
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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.
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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...
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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.
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Specific blood cell populations can be enriched/purified for Nucleofection™ by MACS™ selection Kits (Miltenyi Biotec GmbH), RosetteSep™ Kits (StemCell Technologies Inc.) or Dynal™ beads (Invitrogen Corporation).
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Antibiotics for selection should be added 24-48 h after Nucleofection®.
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We recommend using negative selection (depletion) because your purified cells are "untouched", not influenced, and strictly not activated.Positive selection may cause increasing amounts of dead cells and could also lead to activation of the cells due...
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The culture medium should be changed every 2-3 days during the selection process.This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.
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