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Can I use the pmaxGFP™ control plasmid with insect cells such as S2 cells or SF9 cells?

The pmaxGFP™ Vector provided in our Nucleofector™ Kits is not expressed in insect cells. We strongly recommend an insect expression vector encoding a fluorescent protein or lacZ reporter as a positive control for your experiments [e.g. Novagen™’s...

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....

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...

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.

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...

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.

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...

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

With the 96-well Shuttle™ Device, 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 easily performed...

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