Zitierungen und Referenzen (13)

Invitrogen™

pBAD TOPO™ TA Expression Kit

Name: pBAD TOPO™ TA Expression Kit
SKU: K430001
Price: 1.416.00 EUR

Das pBAD TOPO™ TA-Expressionskit wurde speziell für die einstufige Klonierung und die regulierte prokaryotische Expression von mit Taq-amplifizierten PCR-Produkten entwickelt.Weitere Informationen

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Katalognummer	Proteinmarkierung	Menge
K430001	His-Tag (6x), V5-Epitop-Tag	20 Reaktionen
K430040	V5-Epitop-Tag	40 Reaktionen

2 Optionen

Katalognummer K430001

Preis (EUR)

1.416,00

Each

Proteinmarkierung:

His-Tag (6x), V5-Epitop-Tag

Menge:

20 Reaktionen

Preis (EUR)

1.416,00

Each

Das pBAD TOPO™ TA-Expressionskit wurde speziell für die einstufige Klonierung und die regulierte prokaryotische Expression von mit Taq-amplifizierten PCR-Produkten entwickelt. Sobald Sie Ihr PCR-Produkt mit TOPO™ kloniert haben, können Sie sofort mit der Proteinexpression beginnen. Einige der praktischen Funktionen des pBAD-TOPO™ Vektors:

• Linearisierter, durch Topoisomerase I aktivierter Vektor für 5-minütiges Klonen von mit Taq amplifizierten PCR-Produkten
• Der araBAD-Promotor für die streng regulierte Expression in E. coli V5-Epitop-Tag für den Nachweis mit einem Anti-V5-Antikörper
• C-Terminaler Polyhistidin-Tag (6xHis) zur Aufreinigung mit Nickel-Chelatharz und zum Nachweis mit Anti-His(C-Term)-Antikörper
• Enterokinase-Spaltstelle zur Entfernung des N-terminalen Signalpeptids

Nur für Forschungszwecke. Nicht zur Verwendung bei diagnostischen Verfahren.

Specifications

Bakterielle AntibiotikaresistenzAmpicillin (AMPR)

Bakterien- oder HefenstammLMG194, TOP10

SpaltungEK (Enterokinase)-Erkennungsstelle

Konstitutives oder induktives SystemInduzierbar

ExpressionsmechanismusZellbasierte Expression

ExpressionssystemE. coli

InduktionsmittelArabinose

ProdukttypTA Expressionskit

Menge20 Reaktionen

Selektionsmittel (eukaryotisch)Keine

VektorpBAD

KlonierungsmethodeTopo™-TA

ProduktlinieTOPO

PromoteraraBAD

ProteinmarkierungHis-Tag (6x), V5-Epitop-Tag

Unit SizeEach

Inhalt und Lagerung

Jedes pBAD TOPO™ TA-Expressionskit enthält zwei Boxen und den LMG194 E. coli-Stich. Die pBAD TOPO™ TA-Box enthält alle für die PCR erforderlichen Reagenzien (außer Taq-Polymerase), einschließlich 200 ng durch Topoisomerase I aktivierten pBAD TOPO™ Vektor, steriles Wasser, dNTPs 10x PCR-Puffer, Salzlösung, Kontroll-Template und -Primer, 20 % L-Arabinose, pBAD-Primer für Sequenzierung oder PCR-Screening und ein Expressionskontrollplasmid. Bei -20°C lagern. Die One Shot™ Box enthält alle für die Transformation erforderlichen Reagenzien, einschließlich 50 µl-Einweg-Aliquoten von TOP10 One Shot™ Chemisch kompetentem E. coli, S.O.C.-Medium und einem superhelikalen Kontrollplasmid. Lagerung von One Shot™ Zellen bei -80 °C. Lagerung des LMG194 E. coli-Stabs bei 2 bis 8 °C. Alle Reagenzien bleiben bei ordnungsgemäßer Lagerung garantiert 6 Monate stabil.

Häufig gestellte Fragen (FAQ)

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

Zitierungen und Referenzen (13)

Zitierungen und Referenzen

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