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What stock concentration of Propidium Iodide does Lonza use for FACS analysis after Nucleofection®?

We use a stock concentration of 100 µg/ml in PBS. This is diluted 1:300 – 1:400 into the sample.

What are the requirements for direct Nucleofection® of mRNA for protein expression?

The mRNA should be capped and polyadenylated. The conditions of Nucleofection® will be the same as for DNA with the particular cell type, i.e. follow the same protocol and use the same program, except one will likely need to add a much higher...

In your optimized Nucleofection® Protocols for MDA-MB-231 and MDA-MB-468, you say to culture the cells without CO2. Why?

The media recommended for these cells is Leibovitz's L-15 medium which does not contain sodium bicarbonate to act as a buffer for the carbonic acid that would normally form in the presence of CO2. Furthermore, if the cells are cultured in Leibovitz's...

Do you have any siRNA Nucleofection® results using concentrations lower than 50nM?

Yes. Please click on the attached Technical Reference Guide: "Designing an RNAi Experiment Using Nucleofection®". On page 3 you can find a table with some examples.

Can I use the Nucleofector® Technology for RNAi applications? How do I start?

Yes. The Nucleofector® Technology can be easily applied for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type specific Optimized Protocol for DNA vectors (cDNA, shRNA or miRNA expressing plasmids) or...

Do you have a protocol for the Nucleofection® of Plasmodium berghei using the Nucleofector® Systems

Nucleofector I/II/2bWe have excellent data for the Nucleofection™ of Plasmodium berghei from the group Chris J. Janse and Andrew P. Waters from the University of Leiden. Their protocol describes a transfection efficiency of 10-3 – 10-4. With this...

Can I order any of the Nucleofector® Kit components separately?

No, we do not offer the components of the Nucleofector® Kits separately. The Nucleofection® Kits are only available as complete kits.

Why do you have different Optimized Protocols for the Nucleofection® of unstimulated and stimulated Human T cells?

We found that the stimulation of T cells not only changes the cells' function but also significantly alters the requirements for successful Nucleofection®. That's why Lonza developed two Optimized Protocols for transfection giving different...

For hepatocytes, is plating density a concern post Nucleofection®?

Yes, the optimized protocols for the standard Nucleofector® recommend the use of 6 well plates. Plating into a 12-well plate will pose a problem as less cells will attach at high densities.

Four hours after Nucleofection®, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection® but by 24 hours post Nucleofection®, morphology should be normal. Remember to perform a fluid change about 4 hours post...

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