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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...

What protocol should I use for Nucleofection™ of patient derived blood samples, e.g. leukemia or lymphoma cells?

We do not have a ready-to-use protocol for Chronic lymphocytic leukemia (CLL) or other blood cell derived cancer cells. The cells often have a chromosomal aberration and show the properties of a cell line. Therefore we suggest to perform an...

What antibiotic concentration should I use for selecting stable transfectants?

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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 gating for...

Is it possible to transfect siRNA into blood cells from NHL patients?

Yes. Twin et al ( Blood 2007 Sep 1;110(5):1631-8.) optimized the conditions with pmax-GFP DNA and got 50-75% transfection efficiency for the patient derived diffuse large B-cell lymphoma (DLBL) and mantle cell lymphoma (MCL) cells. This set up was...

How much DNA should I use in my Nucleofection™ Reaction to obtain stable clones?

You should use the amount of DNA recommended in the optimized protocol for transient transfection. If obtaining a very high number of clones is important for your experiments, you can try increasing the amount of DNA. For further tips see our...

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...

How should I purify my DNA for Nucleofection™ of neurons?

The quality and the concentration of DNA used for Nucleofection™ in general plays a central role for the efficiency of gene transfer. We strongly recommend using endotoxin free prepared DNA. Endotoxin free Kits are available from several suppliers....

I've noticed that duration of the Nucleofector™ Program seems to change even when I haven't changed the program I am using. Why?

The duration time of a program is dependent on the number of the program. The duration is also dependent on how long the Nucleofector™ Device has been turned on as well as the number of Nucleofections you have done in the last couple of minutes. When...

What is the lowest amount of DNA which can be used for transfection? Do I need a DNA carrier for low DNA amounts?

The recommended amount of DNA per 100 µl reaction is 0.5µg-5 µg. No carrier is needed.

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