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Four hours after Nucleofection®, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection® but by 24 hours post Nucleofection®, morphology should be normal. Remember to perform a fluid change about 4 hours post...

What are the critical steps for successful Nucleofection® of monocytic cell lines like THP-1, HL60 and U-937?

To achieve optimal results, we strongly recommend following the Optimized Protocols in regard to cell culture conditions (medium, splitting cycle, density before Nucleofection), DNA amount and purity.Please also be sure not to exceed 90xg when...

When nucleofecting cancer cell lines, do I need to be concerned about passage number?

For the most efficient gene transfer, we recommend using cells that are in logarithmic growth phase and at a passage number of 10-15 (from the time of thaw). This is because some cell lines differentiate and change their features after many...

Why do plasmids which contain IRES sequences often have lower transfection efficiency?

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...

Why do plasmids that contain LTR sequences often have lower transfection efficiency?

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...

Why am I not able to detect my fluorescent labeled siRNA (e.g. FITC) after 24 or 48 hours post-Nucleofection?

Fluorescently labeled siRNA duplexes can be used to analyze transfection efficiency by fluorescence microscopy or flow cytometry. However, FITC, Rhodamine, or Alexa-488 labeled siRNA oligos should be analyzed 0.5-3 hours post-Nucleofection™. Cy-5...

Is it more difficult to detect DsRed by FACS than GFP?

Yes, DsRed is much less bright than GFP and is more difficult to detect by FACS and microscopy because it bleaches quickly.

Can I use the pmaxGFP™ control plasmid with insect cells such as S2 cells or SF9 cells?

The pmaxGFP™ Vector provided in our Nucleofector® Kits is not expressed in insect cells. We strongly recommend an insect expression vector encoding a fluorescent protein or lacZ reporter as a positive control for your experiments [e.g. Novagen™’s...

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...

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