pBAD TOPO™ TA Expression Kit

Citations & References (2)

Invitrogen™

pBAD TOPO™ TA Expression Kit

Name: pBAD TOPO™ TA Expression Kit
SKU: K430040

The pBAD TOPO™ TA Expression Kit is specifically designed for one-step cloning and regulated prokaryotic expression of Taq-amplified PCR products.Read more

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Catalog Number	Protein Tag	Quantity
K430040	V5 Epitope Tag	40 Reactions
K430001	His Tag (6x), V5 Epitope Tag	20 Reactions

2 Options

Catalog number K430040

Price (BRL)

Protein Tag:

V5 Epitope Tag

Quantity:

40 Reactions

The pBAD TOPO™ TA Expression Kit is specifically designed for one-step cloning and regulated prokaryotic expression of Taq-amplified PCR products. Once you've TOPO™ Cloned your PCR product, you can go straight to protein expression. Some of the convenient features of the pBAD-TOPO™ vector include:

• Linearized, topoisomerase I-activated vector for 5-minute cloning of Taqamplified PCR products
• The araBAD promoter for tightly regulated expression in E. coli V5 epitope tag for detection with an Anti-V5 Antibody
• C-terminal polyhistidine (6xHis) tag for purification using nickel-chelating resin and detection with an Anti-His(C-term) Antibody
• Enterokinase cleavage site for removal of N-terminal leader peptide

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Antibiotic Resistance BacterialAmpicillin (AmpR)

Bacterial or Yeast StrainLMG194, TOP10

CleavageEK (Enterokinase) Recognition Site

Constitutive or Inducible SystemInducible

Expression MechanismCell-Based Expression

Expression SystemE. coli

Inducing AgentArabinose

Product TypeTA Expression Kit

Quantity40 Reactions

Selection Agent (Eukaryotic)None

VectorpBAD

Cloning MethodTOPO-TA

Product LineTOPO

PromoteraraBAD

Protein TagV5 Epitope Tag

Unit SizeEach

Contents & Storage

Each pBAD TOPO™ TA Expression Kit contains two boxes and the LMG194 E. coli stab. The pBAD TOPO™ TA box contains all of the reagents necessary for PCR (except Taq polymerase), including 200 ng of topoisomerase I-activated pBAD-TOPO™ vector, sterile water, dNTPs, 10X PCR buffer, salt solution, control template and primers, 20% L-arabinose, pBAD primers for sequencing or PCR screening, and an expression control plasmid. Store at -20°C. The One Shot™ box contains all of the reagents necessary for transformation, including single-use 50 μl aliquots of One Shot™ TOP10 Chemically Competent E. coli, S.O.C. medium, and a supercoiled control plasmid. Store One Shot™ cells at -80°C. Store the LMG194 E. coli stab at at 2 - 8°C. All reagents are guaranteed stable for 6 months when properly stored.

Frequently asked questions (FAQs)

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, 40 Reactions FAQs

Citations & References (2)

Citations & References

Abstract

Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from Mycobacterium leprae.

Authors: Reeve Ian; Hummell David; Nelson Nathan; Voss John;

Journal:Proc Natl Acad Sci U S A

PubMed ID:12077319

'It has long been recognized that the pathogenicity of a broad range of intracellular parasites depends on the availability of transition metal ions, especially iron. Nramp1 (natural resistance-associated macrophage protein 1), a proton-coupled divalent metal ion transporter, has been identified as a controlling factor in the resistance or susceptibility to ... More

Ultrafast ligand rebinding in the heme domain of the oxygen sensors FixL and Dos: General regulatory implications for heme-based sensors.

Authors: Liebl Ursula; Bouzhir-Sima Latifa; Negrerie Michel; Martin Jean-Louis; Vos Marten H.;

Journal:Proc Natl Acad Sci U S A

PubMed ID:12271121

Heme-based oxygen sensors are part of ligand-specific two-component regulatory systems, which have both a relatively low oxygen affinity and a low oxygen-binding rate. To get insight into the dynamical aspects underlying these features and the ligand specificity of the signal transduction from the heme sensor domain, we used femtosecond spectroscopy ... More