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

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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.

Answer Id: 3870

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Can N-terminal or C-terminal tags be attached to a Gateway® Entry clone?

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To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO® adapted Gateway® entry vectors. All TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.

To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.

Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.

Answer Id: 3950

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