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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...

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...

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 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...

How can I remove the cells from the 100 uL Nucleocuvette® Vessel after Nucleofection®? Is there any alternative?

We recommend using the plastic single use pipettes provided in the kits. These pipettes prevent any damage to the cells and ensure a good cell recovery.

Is your pmaxGFP control vector supplied with the Nucleofector® Kits suitable for stable expression?

No. This vector is only supplied in our Nucleofector® Kits as a positive control and cannot be used for selection because it does not contain a mammalian selection marker. The only resistance gene expressed by this vector is kanamycin which is...

Do you have any recommended methods for detaching human monocytes from the plate for assaying or before Nucleofection®?

We have had good results by incubating the cells in ice cold PBS for 10 minutes and then rinsing the plates. Alternatively, the cells can be detached without a medium change by gently pipetting the cell suspension up and down. Our experience has also...
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