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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.

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 for...

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.

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...

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...

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....

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. When...

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.

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.

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