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

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

Does your Nucleofector® Solutions contain anything that would inhibit attachment of adherent cells?

No. In many cases where decreased attachment is a problem, the cause is inactivated trypsin. Unless the trypsin is inactivated with trypsin inhibitor or media containg BSA or serum, it will continue to degrade the cells and ultimately decrease cell...

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

The molecular weight of the protein is about 27kDa.

What are your recommendations for minimum and maximum cell numbers for Nucleofection™ ?

The recommended cell number will vary depending on which Optimized protocol is being used. In general, using less than 2x10exp5cells per reaction causes a major increase in cell mortality. For some cell lines we have tried cell numbers up to...

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...

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...

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 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 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 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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