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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...

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 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...

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

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

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....

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

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

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....

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™ Device 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.
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