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Is it possible to get neomycin resistant clones after Nucleofection with pmaxGFP?

The pmaxGFP control DNA is not suitable for making stable clones because it lacks a selectable marker for mammalian cells. The kanamycin resistance expressed by this plasmid is only suitable for bacterial selection.

Where can I find the expiration dates for the Nucleofector Kit and the Nucleofector Solution?

The expiration dates are located on the outside label of the kit box and on the label of the solution box.

Is your pmaxGFP control vector supplied with the Nucleofector Kits suitable for stable expression?

No. This vector is only supplied in our Nucleofector™ Kits as a positive control and cannot be used for selection because it does not contain a mammalian selection marker. The only resistance gene expressed by this vector is kanamycin which is only...

My Nucleofector Solution was frozen. Is this a problem?

As long as the supplement was not added to the Nucleofector™ Solution, then there is no risk of any damage to the solutions. Even long-term storage of several months did not alter the performance of the Nucleofector™ Solution. However, if the...

Do you have any recommended methods for detaching human monocytes from the plate for assaying or before Nucleofection?

We have had good results by incubating the cells in ice cold PBS for 10 minutes and then rinsing the plates. Alternatively, the cells can be detached without a medium change by gently pipetting the cell suspension up and down. Our experience has also...

I have a GFP construct that I use and usually get anywhere from 50-80% GFP positive cells using FACS analysis while I get only 30-40% using a GFP-NFAT fusion protein driven by the same promoter. Do you have any suggestions?

The Amaxa™ Nucleofection™ Conditions are optimised for each cell type, the solutions and programs are cell type dependent and not vector dependant. The expression of the fusion protein depends on the localization of the protein transcription,...

I currently work with some adherent cell lines, such as 293, which are grown in DMEM media. I have noticed that the mortality seems to be high after Nucleofection. Do you have any recommendations?

For some adherent cell lines we strongly recommend using RPMI instead of DMEM medium. As described in culture conditions of the cell line optimization kit protocol as well as the NIH3T3 (DSMZ) protocol, using RPMI instead of DMEM as "transfer and...

How do you recommend that we isolate splenic lymphocytes from mouse spleens for Nucleofection? Is erythrocyte lysis required?

For the preparation of mouse spleen cells we recommend cutting the spleen once and passing the tissue through a 100µM cell strainer or steel mesh using a plunger. The cells are flushed into a petri dish containing PBS. In order to remove fat, cell...

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 recommend positive selection or depletion for the purification of Human monocytes for Nucleofection?

We only recommend the depletion method. For Example, the RosetteSep™ Isolation Kit for human monocytes [Stem Cell Technologies, Cat. No 15028]. The advantage of depletion is that the monocytes are left untouched by antibodies during the process.
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