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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...

Are your Nucleofector® Solutions checked for RNase activity during your QC process?

Yes. We do test the Solutions with a real time RT-PCR based assay. This assay basically measures the quantity of an RNA template after incubation in our solution. In case there is RNAse contamination the template would be digested which would 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...

What kind of buffer do you recommend for resuspending my siRNA?

Please use the buffer that is recommended by your siRNA manufacturer.

If I use Miltenyi paramagnetic beads for positive selection of cells, i.e. beads remain on the cell surface, does that influence Nucleofection® results?

No, it seems that the beads bound to the cell surface do not have any influence on Nucleofection®. Larger magnetic beads (i.e. > 1µm) may disturb the Nucleofection® process and negatively influence cell viability and efficiency.

What stock concentration of Propidium Iodide does Lonza use for FACS analysis after Nucleofection®?

We use a stock concentration of 100 µg/ml in PBS. This is diluted 1:300 – 1:400 into the sample.

What is the proof that DNA enters the nucleus during Nucleofection®?

There are two facts that prove that the DNA is directly transported into the nucleus: 1.) Using the Nucleofector® Technology, transfection of non-dividing cells is possible. With conventional non-viral transfection methods cell division is a...

What are the requirements for direct Nucleofection® of mRNA for protein expression?

The mRNA should be capped and polyadenylated. The conditions of Nucleofection® will be the same as for DNA with the particular cell type, i.e. follow the same protocol and use the same program, except one will likely need to add a much higher...
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