DES™ TOPO™ TA Expression Kit

The DES™ TOPO™ TA Expression Kit offers one-step cloning of PCR products directly into an inducible DES™ expression vector. TheRead more

Catalog Number	Quantity
K412501	20 reactions

Catalog number K412501

Price (BRL)

Quantity:

20 reactions

The DES™ TOPO™ TA Expression Kit offers one-step cloning of PCR products directly into an inducible DES™ expression vector. The kit uses the linearized, topoisomerase I-activated pMT/V5-His-TOPO™ vector for fast and easy cloning and subsequent high-level expression. Topoisomerase activation of this vector allows PCR products to be ligated in just 5 minutes on your bench top to produce >85%recombinants. The pMT/V5-His-TOPO™ vector has all of the features of the pMT/V5-His vector to simplify protein expression, detection, and purification of recombinant proteins in Drosophila S2 cells.

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

Specifications

Inducing AgentCopper Sulfate

Product TypeTA Expression Kit

Quantity20 reactions

VectorpMT, TOPO-TA Cloning Vectors

Cloning MethodTOPO-TA

Product LineDES, TOPO

PromoterMT

Protein TagHis Tag (6x), V5 Epitope Tag

Unit Size20 reactions

Contents & Storage

Each DES™ TOPO™ TA Expression Kit contains two boxes. The DES™ TOPO™ TA box contains 200 ng of linearized and topoisomerase-activated pMT/V5-His- TOPO™ vector, dNTPs, salt solution, control PCR template and primers, forward and reverse primers for sequencing and PCR screening, and a control
expression plasmid (pMT/lacZ). Store at -20°C. The One Shot™ box contains high-efficiency, single-use 50 μl aliquots of competent TOP10 E. coli cells, S.O.C. medium, and pUC19 supercoiled plasmid control. Store at -80°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.

Do I need to include a Kozak sequence for expression of recombinant proteins in insect cells?

While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:

- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
- Biochim Biophys Acta 1260(1):14-20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149-153.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

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

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

View all DES™ TOPO™ TA Expression Kit FAQs

Citations & References (1)

Citations & References

Abstract

Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium.

Authors:MacPherson MR, Lohmann SM, Davies SA,

Journal:J Biol Chem

PubMed ID:15218025

'cGMP-dependent protein kinase (cGK) forms encoded by the dg2 (for) gene are implicated in behavior and epithelial transport in Drosophila melanogaster. Here, we provide the first biochemical characterization and cellular localization of cGKs encoded by the major transcripts of dg2: dg2P1 and dg2P2. cGMP stimulates kinase activity of DG2P1 (EC(50): ... More