pBAD TOPO™ TA Expression Kit

Citas y referencias (13)

Invitrogen™

pBAD TOPO™ TA Expression Kit

Name: pBAD TOPO™ TA Expression Kit
SKU: K430001

El kit de expresión pBAD TOPO™ TA está diseñado específicamente para la clonación en un solo paso y la expresiónMás información

Cambiar vista

Número de catálogo	Etiqueta de proteína	Cantidad
K430001	Etiqueta His (6x), Etiqueta de epítopo V5	20 reacciones
K430040	Etiqueta de epítopo V5	40 reacciones

2 opciones

Número de catálogo K430001

Precio (MXN)

Etiqueta de proteína:

Etiqueta His (6x), Etiqueta de epítopo V5

Cantidad:

20 reacciones

El kit de expresión pBAD TOPO™ TA está diseñado específicamente para la clonación en un solo paso y la expresión procariótica regulada de productos PCR amplificados con Taq. Una vez que TOPO™ haya clonado su producto de PCR, puede ir directamente a la expresión de proteínas. Algunas de las prácticas características del vector pBAD-TOPO™ incluyen:

• Vector linealizado activado por topoisomerasa I para la clonación de 5 minutos de productos de PCR amplificados con Taq
• Promotor araBAD para una expresión fuertemente regulada en la etiqueta de epítopo E. coli V5 para la detección con un anticuerpo anti-V5
• Etiqueta de polihistidina de terminal C (6xHis) para la purificación con resina quelante de níquel y la detección con un anticuerpo anti-His (terminal C)
• Sitio de escisión de enteroquinasa para la eliminación del péptido líder de terminal N

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

Especificaciones

Resistencia bacteriana a los antibióticosAmpicilina (AMPR)

Cepa bacteriana o de levaduraLMG194, TOP10

HendiduraSitio de reconocimiento de EK (enterocinasa)

Sistema constitutivo o inducibleInducible

Mecanismo de expresiónExpresión basada en células

Sistema de expresiónE. coli

Agente inductorArabinosa

Tipo de productoKit de expresión TA

Cantidad20 reacciones

Agente de selección (eucariótico)Ninguno

VectorpBAD

Método de clonaciónTOPO-TA

Línea de productosTOPO

PromotoraraBAD

Etiqueta de proteínaEtiqueta His (6x), Etiqueta de epítopo V5

Unit SizeEach

Contenido y almacenamiento

Cada kit de expresión pBAD TOPO™ TA contiene dos cajas y el medio E. coli LMG194. La caja pBAD TOPO™ TA contiene todos los reactivos necesarios para PCR (excepto polimerasa Taq), entre los que se incluyen 200 ng de vector pBAD-TOPO™ activado por topoisomerasa I, agua estéril, dNTP, tampón 10X para PCR, solución salina, plantilla de control y cebadores, L-arabinosa al 20%, cebadores pBAD para secuenciación o cribado por PCR y un plásmido de control de expresión. Conservar a -20°C. La caja One Shot™ contiene todos los reactivos necesarios para la transformación, incluidas alícuotas de un solo uso de 50 μl de E. coli One Shot™ TOP10 químicamente competente, medio S.O.C. y un plásmido de control superenrollado. Conservar las células One Shot™ a -80 °C. Conservar el medio E. coli LMG194 entre 2 y 8°C. Se garantiza la estabilidad de todos los reactivos durante 6 meses si se almacenan correctamente.

Preguntas frecuentes

Can I store my competent E. coli in liquid nitrogen?

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

How should I store my competent E. coli?

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

How much L-arabinose should I use to induce expression with the pBAD expression system?

While the amount of L-arabinose can vary depending on your expression experiment, we suggest performing a pilot expression experiment with varying amounts of L-arabinose from 0.00002% to 0.2%.

Should I use TOP10 cells or the LMG194 E. coli strain you offer for expression with my pBAD system?

Top10

Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.

Disdvantages:
- This strain is not protease-deficient. Therefore, the protein may be degraded.

LMG194

Advantages:
- Grows well in minimal media, except M9.
-Have to transform the plasmid into the cells just for expression.
-RM medium with glucose to ensure low basal level of protein.

Disadvantages:
- Not protease-deficient. Therefore, the protein may be degraded.
- The glycerol stock may not be stable because this cell strain is not recA- or endA-.

What competent cells do you recommend I use for expression with my pBAD expression system?

We recommend using a competent cell strain that is araBADC- and araEFGH+, allowing transportation of L-arabinose, but not metabolizing it. This is important for expression studies, as the level of L-arabinose will be constant inside the cell and will not decrease over time. We offer our TOP10 competent cells, or our LMG194 E. coli strain.

View all pBAD TOPO™ TA Expression Kit, 20 Reactions FAQs

Citations & References (13)

Citations & References

Abstract

LuxS is required for persistent pneumococcal carriage and expression of virulence and biosynthesis genes.

Authors:Joyce EA, Kawale A, Censini S, Kim CC, Covacci A, Falkow S,

Journal:Infect Immun

PubMed ID:15102809

'Streptococcus pneumoniae causes several diseases, including otitis media, pneumonia, and meningitis. Although little is known about the regulation of or how individual pneumococcal factors contribute to these disease states, there is evidence suggesting that some factors are regulated by a cell-density-dependent mechanism (quorum sensing). Quorum sensing allows bacteria to couple ... More

Identification of a novel maturation mechanism and restricted substrate specificity for the SspB cysteine protease of Staphylococcus aureus.

Authors:Massimi I, Park E, Rice K, Muller-Esterl W, Sauder D, McGavin MJ,

Journal:J Biol Chem

PubMed ID:12207024

'The SspB cysteine protease of Staphylococcus aureus is expressed in an operon, flanked by the sspA serine protease, and sspC, encoding a 12.9-kDa protein of unknown function. SspB was expressed as a 40-kDa prepropeptide pSspB, which did not undergo autocatalytic maturation. Activity of pSspB was reduced compared with 22-kDa mature ... More

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote giardia lamblia.

Authors:Bunjun S, Stathopoulos C, Graham D, Min B, Kitabatake M, Wang AL, Wang CC, Vivares CP, Weiss LM, Soll D

Journal:Proc Natl Acad Sci U S A

PubMed ID:11078517

'Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per ... More

RNA stem-loop enhanced expression of previously non-expressible genes.

Authors:Paulus M, Haslbeck M, Watzele M,

Journal:Nucleic Acids Res

PubMed ID:15163763

The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. ... More

Shielding of the A1 Domain by the D'D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V.

Authors:Ulrichts H, Udvardy M, Lenting PJ, Pareyn I, Vandeputte N, Vanhoorelbeke K, Deckmyn H,

Journal:J Biol Chem

PubMed ID:16373331

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 ... More

View all pBAD TOPO™ TA Expression Kit, 20 Reactions citations