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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...

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...

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 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 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...

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 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.

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 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...

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.
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