Vivid Colors™ pcDNA™6.2/N-EmGFP-DEST Vector - FAQs

Does EmGFP have the F64L mutation and what effect does this mutation have?

The F64L mutation in EmGFP was purported to render better folding properties to the EmGFP protein at 37 degrees C. However, in-house studies have shown that this mutation is not necessary. This mutation is present in EmGFP (and BFP) in the pRSET vectors. The EmGFP in the pcDNA6.2 DEST vectors, pcDNA6.2 GW/TOPO vectors and plenti6.2 GW vector does not contain this mutation.

Now that Thermo Fisher Scientific is selling the Vivid Colors Aequorea victoria fluorescent protein vectors and Clontech is not, have there been any changes to the licensing requirements?

Use of our Vivid Colors fluorescent protein vectors is described under the Limited Use Label Licenses accompanying the product. For use in research at academic or government (non-profit) agencies, the purchase of the product (as a catalog item) from Thermo Fisher Scientific is sufficient; no further licenses are required. For use in research at a commercial (for profit) entity, a commercial-use license is necessary for Aequorea victoria derived proteins; this applies to EmGFP, YFP, CFP and BFP. For any questions related to licensing, please send an e-mail to outlicensing@lifetech.com

The nomenclature for your Vivid Colors fluorescent protein-tagged vectors seems to be different from Clontech's nomenclature. Why is this?

Clontech named their BD Living Colors-fusion vectors with reference to the location of the ORF in the fusion construct. For example, to fuse an ORF with a C-terminal fluorescent protein tag, a Clontech N-series vector (N-terminal Fusion vector) was used. Our nomenclature is exactly the opposite. This was done to be consistent with the standardized nomenclature we have been using with our expression vector products for many years. For example, our Vivid Colors pcDNA6.2/C-EmGFP-DEST is a vector suitable for Gateway cloning an ORF upstream of an EmGFP-tag. Likewise, the Vivid Colors pcDNA6.2/N-YFP-DEST is a vector suitable for Gateway cloning an ORF downstream of a YFP-tag. So, our Vivid Colors-fusion vectors are named with specific reference to the location of the fluorescent protein tag in the fusion construct.

Can I use E. coli containing an F' plasmid, such as Top 10F', to propagate a vector with the ccdB gene?

While the F' plasmid does contain the ccdA gene that can inhibit or reduce the toxicity of the ccdB gene product, the ccdA expression level is likely to be too low, or inhibition may not be complete, and the bacteria would still be exposed to the ccdB gene product and thus not grow. Therefore, bacterial strains containing the F' plasmid are not recommended as hosts for propagation of ccdB containing vectors.

For propagation of Gateway vectors containing ccdB, we recommend the One Shot ccdB Survival 2 T1R Competent Cells (A10460), which were specifically designed for that purpose. However, please note that these cells are not validated for propagation of other ccdB-containing vectors like the older pZErO plasmids, and in most cases they are not expected to work due to very high levels of ccdB protein expressed in those vectors.