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Lonza recommends using PEG purified DNA for Nucleofection of DC's and macrophages. Is it necessary to perform the PEG purification step for the positive control plasmid pmaxGFP supplied in each Nucleofector Kit?

No, it is not. The PEG purification step removes additional endotoxin from the DNA preparation. The pmaxGFP reporter plasmid supplied in each kit is highly purified and endotoxin levels are <0.01E.U./µg DNA (which means <1pg endotoxin/µg DNA).

What selection markers can I use for stable transfections?

The most commonly used marker is the neomycin phosphotransferase (neo) gene that confers resistance to G418 to eukaryotic cells. Other markers are Puromycin, Hygromycin, Zeocin, or the HPRT gene that can be used in HPRT-deficient cells.

DNA-purity and Nucleofection: Can low A260:A280 ratios lead to both reduced transfection efficiency and cell viability?

Yes. To check the quality of your DNA we strongly recommend determining the A260:A280 ratio. It should be ideally 1.8 - 2.0 for a good DNA preparation, but least 1.6.

Can nicked DNA lead to reduced transfection efficiency?

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Compare undigested plasmid to plasmid DNA digested with a suitable single cut restriction enzyme to linearize. Supercoiled plasmid will run faster than...

How often should I change the medium during selection of stable clones after Nucleofection?

Every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.

Can I use Nucleofection to create a stable knockdown in a cancer cell line?

Yes. There are several publications available showing stable transfection of an shRNA vector: Cai S et al. (2006) Nat Genet 38:1278-88 (D10.G4.1 cells) Guo F et al. (2005) Cancer Res 65:10536-44 (K562) Hideshima T (2006) Blood 107: 4053-62 (U937)...

How many stable clones will I get from one transfection?

This depends on the respective cell type (transfection efficiency, viability and integration frequency). If it is important to you to know the frequency of stable integrants before carrying out your actual experiments, the frequency should be...

What protocol should I use for Nucleofection of patient derived blood samples, e.g. leukemia or lymphoma cells?

We do not have a ready-to-use protocol for Chronic lymphocytic leukemia (CLL) or other blood cell derived cancer cells. The cells often have a chromosomal aberration and show the properties of a cell line. Therefore we suggest to perform an...

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.