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Do you have any information regarding differentiation of mouse ES cells after Nucleofection® with large plasmids?

It is unlikely that plasmid size would affect in vitro differentiation capability. Gene targeting constructs are often as large as 20 kb and if treated well the ES cells are perfectly capable of generating germ line chimaeras afterwards (indicating...

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...

Does the Mouse T cell protocol work for stimulated mouse T cells?

Yes. Stimulate the cells with anti CD3 and anti CD28 antibody for 24 hours. Use the optimized protocol. Often times, this will result in higher transfection efficiency (60-70% is possible). Alternatively, you can stimulate with ConA and IL-2 for 2-3...

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 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...

I've noticed that duration of the Nucleofection® 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® System has been turned on as well as the number of Nucleofections you have done in the last couple of...

Can I apply more than one program (transfection condition) per plate with the 96-well Shuttle® System?

With the 96-well Shuttle® system, it is possible to apply up to 96 different programs (transfection conditions) per 96-well Nucleocuvette® Plate. Therefore, optimization of Nucleofection® Conditions for a particular cell line can be...

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.

Can I use larger cuvettes for my Nucleofection® Reaction? Can I use the cuvettes more than once?

No. The electrical parameters provided by the Nucleofector® System are optimized for the cuvettes contained in the Nucleofector® Kits. The cuvettes are single-use only. Using the cuvettes more than once will result in higher cell death and lower...
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