One Shot® TOP10/P3 Chemically Competent E. coli -"DISCONTINUED" - FAQs

The pcDNA1 vector, pcDNA1.1 vector, pcDM8 vector and pcDNA1/Neo vector do not contain an antibiotic resistance gene for selection inE.coli. Instead, they contain a tRNA suppressor F gene (supF) for E. coli selection. To take advantage of this selection, you will have to propagate the plasmid in specific E.coli strains harboring the plasmid P3. P3 is a low-copy 60 kb episomal plasmid which encodes the kanamycin resistance gene, and it also contains amber mutants of the tetracyline and ampicillin resistance genes. Strains that harbor P3 alone are resistant to kanamycin, but sensitive to both tetracycline and ampicillin - however, the amber mutations are reversed in the presence of the supressor F tRNA, encoded by the supF gene. Thus, when E. coli carrying the P3 plasmid are transformed with supF plasmids (e.g. pcDNA1 vector and pCDM8 vector), they are rendered resistant to both tetracycline and ampicillin by suppression of the amber mutations.

Please note: Amber mutations are simple point mutations and the rate of spontaneous reversion of the mutations on the P3 episome is relatively high. To reduce the chance of background colonies, it is highly recommended to select for both tetracycline and ampicillin resistance - i.e. add both tetracycline and ampicillin to your selection plates.

To propagate these plasmids for transfection, we recommend the One Shot TOP10/P3 Chemically Competent E. coli (Cat. No. C5050-03).  You can find more information in the product manual.

The P3 plasmid is approximately 60 kb and usually is present at only 1 or 2 copies per cell. It is similar in properties to the F' episome. It can be recovered along with your vector of interest in a plasmid prep and might be detectable on a gel. The very large plasmid would be seen high on the gel near the wells.

MC1061/P3 is the strain that was historically used for most supF selection applications. It is referenced by Brian Seed in his library construction paper.

For library transformations, we do still recommend MC1061/P3 because of its slightly higher transformation efficiency when transforming pcDNA1.1. However, for propagating plasmid for transfection, we strongly recommend using the TOP10/P3 instead, which is endA- and yields much cleaner plasmid DNA in purification. DNA prepared from MC1061 cells is typically not suitable for transfection.