One Shot™ TOP10/P3 Chemically Competent E. coli

La E. coli TOP10/P3 permite una preparación de plásmido de alta calidad de pCDM8, pcDNA™1.1 o cualquier otro vector conMás información

Número de catálogo	Cantidad
C505003	21 x 50 μl

Número de catálogo C505003

Precio (CLP)

Cantidad:

21 x 50 μl

La E. coli TOP10/P3 permite una preparación de plásmido de alta calidad de pCDM8, pcDNA™1.1 o cualquier otro vector con supF. Las características de la cepa son:

• Plásmido P3 para la selección de supF con plásmidos
• Gen endA1 mutado para una calidad aumentada de preparaciones de ADN de plásmido

Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.

Especificaciones

Resistencia bacteriana a los antibióticosYes (Streptomycin, Kanamycin, Ampicillin, Tetracycline)

Tramado azul/blancoSí

Clonación de ADN metiladoSí

Clonación de ADN inestableNo es adecuado para clonar ADN inestable

Contiene el episoma F'Carece de episoma F'

Compatibilidad de alto rendimientoNo compatible con alto rendimiento (manual)

Mejora la calidad de los plásmidosSí

Preparación de ADN no metiladoNo es adecuado para preparar ADN no metilado

Línea de productosOne Shot

Tipo de productoCélula competente

Cantidad21 x 50 μl

Reduce la recombinaciónSí

Condiciones de envíoDry Ice

Resistente al fago T1 (tonA)No

Nivel de eficiencia de transformaciónEficacia media (10^8-10^9 ufc⁄µ g)

FormatoTubo

EspecieE. coli

Unit SizeEach

Contenido y almacenamiento

• One Shot TOP10/P3 Chemically Competent E. coli (21 x 50 μL)
Store Competent Cells at –80°C.

• pUC19 Plasmid (50 μL)
Store pUC19 plasmid at –20°C.

S.O.C. Medium (6 mL)
Store S.O.C. Medium at 4°C or at room temperature

Preguntas frecuentes

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. pcDNA1 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.

What is the reason for the nupG mutation in the genotypes for TOP10- and DH10B-related E. coli strains?

nupG is a mutation for the transport of nucleosides. The nupG site is next to endA on the chromosome, and when endA was mutated by transposon insertion, the nupG site was unintentionally mutated as well. There are no apparent effects of this mutation on cell function or growth.

References: 1) Nghiem, Y. et al. PNAS 85: 2709-2713. 2) Westh Hansen, S.V. et al. Eur. J. Biochem. 168: 385-391.

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.

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 pcDNA1 or pcDNA1.1)?

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 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.