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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 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...

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...

Are your Nucleofector Solutions checked for RNase activity during your QC process?

Yes. We do test the Solutions with a real time RT-PCR based assay. This assay basically measures the quantity of an RNA template after incubation in our solution. In case there is RNAse contamination the template would be digested which would be...

Is it possible to transfect siRNA into blood cells from NHL patients?

Yes. Twin et al ( Blood 2007 Sep 1;110(5):1631-8.) optimized the conditions with pmax-GFP DNA and got 50-75% transfection efficiency for the patient derived diffuse large B-cell lymphoma (DLBL) and mantle cell lymphoma (MCL) cells. This set up was...

What kind of buffer do you recommend for resuspending my siRNA?

Please use the buffer that is recommended by your siRNA manufacturer.

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

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

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 is the lowest amount of Human Hepatocytes that can be used with the 96-well Shuttle System or the 4D-Nucleofector X-Unit?

Human hepatocytes do not like to be cultured and transfected at low densities. Therefore, using less than 100,000 cells per Nucleofection™ is not recommended.
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