What is the mechanism for supF selection in E. coli carrying the P3 plasmid? And what is meant by the "amber" notation in the genotype - are these cells resistant to tetracycline and ampicillin?
E. coli harboring the plasmid P3 enable the selection and maintenance of plasmids that encode the tRNA suppressor F gene (supF). P3, a low-copy 60 kb episomal plasmid, encodes the kanamycin resistance gene as well as amber mutants of the tetracyline and ampicillin resistance genes. The amber mutations are point mutations in the resistance markers which inactivate the expression of resistance in the strain unless the tRNA supressor F (supF) is present. Therefore, strains that harbor P3 alone are resistant to kanamycin, but sensitive to both tetracycline and ampicillin. But when the E. coli carrying the P3 plasmid are transformed with supF-containing plasmids (e.g. pcDNA™1 and pCDM8), they are rendered resistant to both tetracycline and ampicillin (as well as kanamycin) by suppression of the amber mutations.
The rate of spontaneous reversion of the amber point mutations on the P3 episome is fairly high, so it is important to select supF clones with both tetracycline and ampicillin resistance to reduce background growth. To avoid enriching for revertants, it is recommended that relatively low concentrations of tetracycline (7.5 to 10 ug/ml) and ampicillin (25 to 40 ug/ml) be used.
Answer Id: 3822
What's the difference between MC1061/P3 and TOP10/P3, and how would I choose one or the other for my supF selection (for example: using pcDNA™1 or pcDNA™1.1)?
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 pcDNA™1.1. 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.
Answer Id: 3352
When examining mini-prep DNA isolated from MC1061/P3, there is a smear extending from high molecular weight to ~5 kb instead of an apparent band at or near where the vector should be. What causes this?
The smear is expected when doing DNA preps in MC1061/P3 because the cells do not carry the endA- mutation. To prevent DNA degradation by endonuclease in plasmid preparations from these cells, an alkaline lysis procedure (i.e. Solutions I,II,III) which contains a phenol:chloroform step that destroys endogenous endonucleases should be used. Many commercially available DNA miniprep kits do not accomplish this efficiently, and boiling the DNA solution after isolation is not effective.
For best DNA preparation results with supF-selection plasmids, we highly recommend the TOP10/P3 One Shot® cells (catalog number C5050-03). This strain yields higher quality and quantity of plasmid DNA due to the presence of both recA- and endA- mutations. For plasmids with antibiotic selection markers rather than supF selection, use one of the more common strains like TOP10, DH5a™ or DH10B™.
Answer Id: 3353
How large is the P3 plasmid in the MC1061/P3 or TOP10/P3 cells, and is it isolated with other plasmids in standard preps?
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.
Answer Id: 3354