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Invitrogen™

pTrcHis2 TOPO™ TA Expression Kit

Los kits de expresión pTrcHis- y pTrcHis2-TOPO™ TA permiten la clonación rápida y eficaz de productos de PCR en unMás información
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Número de catálogoCantidad
K44000120 reacciones
Número de catálogo K440001
Precio (MXN)
-
Cantidad:
20 reacciones
Los kits de expresión pTrcHis- y pTrcHis2-TOPO™ TA permiten la clonación rápida y eficaz de productos de PCR en un vector de expresión procariótica para una expresión regulada de alto nivel del promotor trc. El vector en cada kit, pTrcHis-TOPO™ o pTrcHis2-TOPO™, incluye un elemento de minicistron para mejorar la eficacia de la traslación de las proteínas eucariotas en E. coli. Ambos vectores se proporcionan linealizados y activados con topoisomerasa I para una clonación TOPO™ de 5 minutos con una eficacia de clonación >85 %. Para simplificar la detección y purificación de proteínas, los vectores pTrcHis-TOPO™ y pTrcHis2-TOPO™ incluyen las siguientes características:

pTrcHis-TOPO™

• Epítopo Xpress™ de terminal N para la detección con un anticuerpo Anti-Xpress™
• Una etiqueta de polihistidina de terminal N (6xHis) para la purificación con resina quelante de níquel y detección con un anticuerpo anti-HisG
• Sitio de escisión de enterocinasa para una eliminación eficaz de las etiquetas de fusión de terminal N

pTrcHis2-TOPO™
• Epítopo c-myc de terminal C para la detección y análisis con un anticuerpo anti-myc
• Etiqueta de polihistidina de terminal C (6xHis) para purificación con resina quelante de níquel y detección con un anticuerpo anti-His (terminal C)
Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.
Especificaciones
Resistencia bacteriana a los antibióticosAmpicilina (AMPR)
Cepa bacteriana o de levaduraTOP10
HendiduraSitio de reconocimiento de EK (enterocinasa)
Sistema constitutivo o inducibleInducible
Mecanismo de expresiónExpresión basada en células
Sistema de expresiónE. coli
Agente inductorIPTG
Tipo de productoKit de expresión TA
Cantidad20 reacciones
Agente de selección (eucariótico)Ninguno
VectorpTrc
Método de clonaciónTOPO-TA
Línea de productosTOPO
PromotorTrc, lacO
Etiqueta de proteínaEtiqueta His (6x), Etiqueta de epítopo c-Myc
Unit SizeEach
Contenido y almacenamiento
Los kits de expresión pTrcHis2-TOPO™ TA contienen dos cajas cada uno. La caja TOPO™ TA contiene 200 ng de vector pTrcHis-TOPO™ o pTrcHis2-TOPO™linealizado activado por topoisomerasa I, agua estéril, dNTP, tampón 10X para PCR, muestra y cebadores de control, cebadores para secuenciación o cribado por PCR y un plásmido de control de expresión. La caja One Shot™ TOP10 contiene veintiuna alícuotas de 50 μl de E. coli TOP10 químicamente competente, medio S.O.C. y un plásmido de control superenrollado. Conservar la caja TOPO TA Cloning™ a -20°C. Conservar la caja One Shot™ a -80°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.

What are the advantages and/or disadvantages of using TOP10, DH5α, or other cloning strains versus BL21 Star (DE3) or BL21 (DE3) cells for expression with the pTrc system?

Top10, DH5α, other cloning strains

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

Disdvantages:
- If GOI is toxic, the cloning step can be difficult.
- These cloning strains are not protease-deficient; therefore, the protein may be degraded.

BL21 Star(DE3) or BL21 (DE3)

Advantages:
- These expression strains are protease-deficient.
- You have to transform the plasmid into an expression strain.
- The glycerol stock may be unstable because these expression strains are not endA- and recA-.
- The (DE3) part is wasted because the promoter does not need T7 RNA polymerase.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What competent cell strain can I use for expression with my pTrc Expression System?

The pTrc promoter is recognized by E. coli RNA polymerase, not T7 polymerase, and therefore can be expressed in any E. coli strain, not just BL21 strains. Therefore, you can use Top10, DH5α, etc. for expression. However, if your gene is toxic, the cloning step can be difficult as there is leakiness in expression.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How does glucose repress the pTrc promoter?

A transcriptional activator protein called CAP (catabolite activator protein) normally binds upstream of the trc promoter and activates transcription. This protein requires cAMP to bind the DNA. Adding glucose to the medium can reduce intracellular cAMP levels. Supplementing LB medium and agar plates with glucose will repress basal level transcription from the trc promoter. We recommend that you include 25 mM, 0.5% glucose in the selection medium to ensure stability of your insert.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all pTrcHis2 TOPO™ TA Expression Kit FAQs

Citations & References (5)

Citations & References
Abstract
Characterization of a Novel Drosophila melanogaster Galectin. EXPRESSION IN DEVELOPING IMMUNE, NEURAL, AND MUSCLE TISSUES.
Authors: Pace Karen E; Lebestky Tim; Hummel Thomas; Arnoux Pascal; Kwan Kent; Baum Linda G;
Journal:J Biol Chem
PubMed ID:11809773
'We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding beta-galactoside sugars. Confirming its identity as a galectin family member, the ... More
Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Transcriptional Activator Rta Is an Oligomeric DNA-Binding Protein That Interacts with Tandem Arrays of Phased A/T-Trinucleotide Motifs.
Authors:Liao W, Tang Y, Kuo YL, Liu BY, Xu CJ, Giam CZ,
Journal:J Virol
PubMed ID:12915555
'Kaposi''s sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) encodes an immediate early transcriptional activator, Rta, which mediates viral reactivation from latency and lytic viral replication. Here we report the purification and characterizations of HHV-8 Rta and its interaction with Rta-responsive DNA elements. The Rta response element (RtaRE) in the promoter ... More
Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica.
Authors:Grose JH, Bergthorsson U, Xu Y, Sterneckert J, Khodaverdian B, Roth JR,
Journal:J Bacteriol
PubMed ID:15968063
'Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and ... More
Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum.
Authors: Kicska Gregory A; Tyler Peter C; Evans Gary B; Furneaux Richard H; Kim Kami; Schramm Vern L;
Journal:J Biol Chem
PubMed ID:11707439
'Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present ... More
Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis.
Authors: Simm Alan M; Higgins Catherine S; Carenbauer Anne L; Crowder Michael W; Bateson John H; Bennett Peter M; Clarke Anthony R; Halford Stephen E; Walsh Timothy R;
Journal:J Biol Chem
PubMed ID:11940588
The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of ... More