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Following Nucleofection, I cannot obtain stable clones from single cells, what could be the reason?

Some cell lines need certain cell densities to proliferate. For those cells you can try using conditioned medium for setting up limiting dilution or culturing single cells together with feeder cells.

How much DNA should I use in my Nucleofection Reaction to obtain stable clones?

You should use the amount of DNA recommended in the optimized protocol for transient transfection. If obtaining a very high number of clones is important for your experiments, you can try increasing the amount of DNA. For further tips see our...

I want to stimulate human primary T-cells with anti-CD3 and anti-CD28. Do you have a detailed protocol for this?

For optimal stimulation of the transfected cells we recommend coating 96-wells plates for stimulation [Nunc Immuno™ Plate C96 Maxi Sorp™, Cat. No. 430 341] with 50 µl of a solution of anti- CD3 antibody [OKt 3; eBioscience, Cat. No. 14-0037-82; final...

Is there a reason why in our Nucleofector Protocols for Primary Human Chondrocytes we include FCS in the pronase and collagenase solutions for preparation of chondrocytes?

Yes, even though this may sound odd since serum is generally known to inactivte proteases. We recommend it in this case, however, because the incubation times are quite long and the cells are stressed by the procedure. The amounts of enzymes should...

Does the GFP sequence in your pmaxGFP control plasmid have a stop codon?

Yes. The coding sequence ends with a stop codon (TGA/UGA).

What antibodies can be used against the GFP produced by pmaxGFP and pmaxFP-Green vectors? What antibodies can be used to detect denatured GFP? What antibodies can be used to detect the RFP expressed by pmaxFP-Red?

Most of the commercial antibodies for GFP do not detect GFP expressed from pmaxGFP or pmaxFP-Green vectors. The origin of the protein expressed by the pmaxGFP control plasmid and pmaxFP-Green is from the green fluorescent protein CopGFP, cloned from...

What is the shelf life of the 96-well Shuttle kits?

The shelf life of the 96-well Shuttle™ Kits is 24 months from date of manufacturing. We guarantee a remaining shelf life of at least 6 months after sending them out of the warehouse.