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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...

Does the 96-well Shuttle™ recognize if the cuvette plate is in backwards?

No, it does not recognize the orientation of the plate. Please make sure that well A1 is positioned in the upper left corner of the retainer.

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...

Do any of the three instruments used in the 96-well Shuttle™ System "go to sleep" or power off automatically?

The energy options of the laptop are set as such that only the screensaver is activated, the harddisk won't turn down and hibernation is disabled. One can change these settings via the system control. However this is not recommended because by...

What is the molecular weight of your control pmaxGFP™ plasmid?

The molecular weight is approximately 2.3x10e6 g/mol based on the average MW of a base pair being 660 g/mol.

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...

What cell number do I need per well when using the 96-well Shuttle™ Device or the 4D-Nucleocuvette™ Stripes?

The cell number is very much dependent on the cell type you use. For high throughput Nucleofection™, we usually start with one fifth of the optimal amount recommended for the standard Nucleofection™ in the 100µl cuvette. Currently the absolute cell...

What is the molecular weight of the GFP protein expressed by your control pmaxGFP™ plasmid?

The molecular weight of the protein is about 27kDa.

What is the amount of DNA needed per well when using the 96-well Shuttle™ Device?

The amount depends on the cell type and on the DNA construct. As a rule of thumb 400 ng of DNA per well should be applied. However, for some cells or constructs even 100 ng may be sufficient.

What is the amount of siRNA needed per Nucleofection™ Reaction?

When performing siRNA-mediated knockdown experiments it is advisable to conduct a dose-response (concentration) analysis to determine the minimum siRNA concentration necessary for sufficient target knockdown on the mRNA, protein, or functional level....

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