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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...

Does the MycoAlert® Assay recognize the whole spectrum of mycoplasma?

The mycoplasma specific metabolism detected by the MycoAlert® Assay is present in all members of the mollicute family (Mycoplasma, Acholeplasma, Entomoplasma and Spiroplasma) except Ureaplasma (which are no usual suspects in cell culture). 95% of all...

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 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 bacteria lead to false positive MycoAlert® Results?

NO - the lysis reagent in the MycoAlert® Assay is not strong enough to lyse bacteria / yeast. Also, the enzymes utilized are not very common in bacteria or yeast – but are universal among the mollicute family. Bacterial contamination is...

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...

Can mycoplasma grow in the absence of cells?

There is evidence to suggest that mycoplasma can survive in the absence of cells, some may even proliferate, but as a rule they are much more viable if cells are present. 

Is it possible to store reconstituted MycoAlert®/MycoAlert® PLUS Reagent and Substrate?

For optimal assay conditions, reconstituted reagent and substrate should be used fresh within 2 hours after reconstitution.During this time they can be kept at room temperature.For the MycoAlert® Kit: Unused, reconstituted components can be aliquoted...

How much sample do you need for accurate results with MycoAlert® Assay? Do you need cells or just supernatant?

The standard protocol calls for taking 2 ml of culture media, spinning down any cells and removing 100 µl of supernatant as sample.Smaller initial aliquots of media may be taken to start with if necessary.

I've noticed that duration of the Nucleofection® 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® System has been turned on as well as the number of Nucleofections you have done in the last couple of...
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