Células compententes One Shot™ ccdB Survival™ 2 T1R
Células compententes One Shot&trade; <i>ccd</i>B Survival&trade; 2 T1<sup>R</sup>
Invitrogen™

Células compententes One Shot™ ccdB Survival™ 2 T1R

Las células competentes químicamente One Shot™ ccdB Survival™ 2 T1R son adecuadas para la propagación de plásmidos que contienen elMás información
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Número de catálogoCantidad
A1046011 x 50 μl
Número de catálogo A10460
Precio (MXN)
-
Cantidad:
11 x 50 μl
Las células competentes químicamente One Shot™ ccdB Survival™ 2 T1R son adecuadas para la propagación de plásmidos que contienen el gen ccdB y se han diseñado para su uso con el sistema de conversión de vectores Gateway™ y para propagar vectores de entrada superhelicoidal, donante y destino Gateway™. La eficacia de transformación es >1 × 109 transformantes/µg de ADN plasmídico. La cepa ccdB Survival™ 2 T1R E. coli deriva de la cepa TOP10. La cepa ccdB Survival™ 2 T1R E. coli ofrece las siguientes características:

• Resistencia al producto del gen ccdB la
• Resistencia al fago T1 y T5 (el fenotipo tonA/fhuA)
• Favorece la preparación de alta producción de ADN plasmídico superenrollado (el fenotipo end A1)
• Reducción de la recombinación inespecífica del ADN clonado (el fenotipo rec A1)

Genotipo: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA:IS2
Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.
Especificaciones
Resistencia bacteriana a los antibióticosYes (Streptomycin)
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
PlásmidoccdB vector propagation
Preparación de ADN no metiladoNo es adecuado para preparar ADN no metilado
Línea de productosOne Shot
Tipo de productoCélula competente
Cantidad11 x 50 μl
Reduce la recombinaciónSí
Condiciones de envíoHielo seco
Resistente al fago T1 (tonA)Sí
Nivel de eficiencia de transformaciónAlta eficacia (> 10^9 ufg⁄µ g)
FormatoTubo
EspecieE. coli
Unit SizeEach
Contenido y almacenamiento
Contiene:
• Células competentes ccdB Survival™2 T1R: 11 viales, 50 µl cada uno
• pUC19 DNA (10 pg/µl): 1 vial, 50 µl
• Medio S.O.C.: 1 frasco, 6 ml

Almacenar las células competentes a -80 °C. Almacene pUC19 DNA a -20 °C. Almacenar el medio S.O.C. a 4 °C o a temperatura ambiente.

Preguntas frecuentes

I just found out that Library Efficiency DB3.1 and One Shot ccdB Survival T1R cells have been discontinued. Do you offer an alternative for propagating ccdB-containing plasmids?

We offer One Shot ccdB Survival 2 T1R cells (Cat. No. A10460) for propagating ccdB-containing plasmids.

What kind of competent cells should I use to propagate Donor vectors and Destination vectors? What cells should I use after the BP or LR recombination reaction?

All Donor vectors and Destination vectors contain the ccdB cell death gene to reduce background of non-recombined BP/LR plasmids. Therefore, growing non-recombined vector requires special cells (One Shot ccdB Survival 2 T1R Competent Cells) which are resistant to the lethal effects of ccdB. On the other hand, general E. coli cloning strains including TOP10 or DH5a may be used for plating the BP or LR reaction, or for propagation and maintenance of recombined Gateway constructs.

I need a competent cell strain that is ccdB resistant. The Library Efficiency DB3.1 cells you offer have been discontinued, as well as the One Shot ccdB Survival T1R chemically competent cells. What do you recommend?

Please use our ccdB Survival 2 T1R competent cell strain.

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.