BaculoDirect™ C-Term Expression Kit

Citations & References (9)

Invitrogen™

BaculoDirect™ C-Term Expression Kit

The BaculoDirect™ Baculovirus Expression System is a powerful and versatile eukaryotic system for high-level protein expression in insect cells. TheRead more

Catalog Number	Quantity
12562013	5 transfections

Catalog number 12562013

Price (EUR)

2.505,00

Add to cart

Quantity:

5 transfections

Price (EUR)

2.505,00

Add to cart

The BaculoDirect™ Baculovirus Expression System is a powerful and versatile eukaryotic system for high-level protein expression in insect cells. The combination of Gateway™ Technology with baculovirus expression makes the BaculoDirect System the fastest and easiest method for production of recombinant baculovirus.

How it works
BaculoDirect™ Linear DNA (the baculovirus genome) is engineered to include attR sites for quick and efficient recombination with a Gateway entry clone. The gene of interest is recombined from the entry clone into the BaculoDirect Linear DNA using a simple, one-hour LR reaction (Figure 1). The resulting reaction mix contains the recombinant baculovirus carrying the gene of interest and is used to transfect insect cells. The need for transforming bacteria and isolating a large bacmid or co-transfecting a transfer vector and linear baculovirus DNA into insect cells is eliminated. As a result, the hands-on time is greatly reduced. Purified baculovirus can be isolated in less than one week.

Gateway linear DNA
BaculoDirect Linear DNA is designed for rapid cloning with a Gateway entry clone and subsequent expression in Sf9 or Sf21 insect cells. The Linear DNA features:

• Strong polyhedrin promoter for high-level expression
• R-sites for efficient recombination with any attL-flanked Gateway entry vector
• TK gene for negative selection using ganciclovir
• C-terminal V5-His tag (BaculoDirect™ C-Term Expression Kit) or N-terminal V5-His tag (BaculoDirect™ N-Term Expression Kit) for detection with anti-V5 antibody and purification with nickel-chelating resin
• TEV protease cleavage site for removal of the V5-His tag following purification (BaculoDirect N-Term Expression Kit)
• LacZ gene, to ensure a pure baculovirus stock is generated, which is replaced by your gene of interest after LR recombinant reaction

Additional materials required, available separately: Gateway entry clone.

A selection guide for choosing the most appropriate Gateway entry vector for your application can be found at: www.thermofisher.com/Gateway.

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

Specifications

Product TypeExpression Kit

Quantity5 transfections

VectorBaculoDirect Vectors

Cloning MethodGateway

Product LineBaculoDirect, Gateway

PromoterPolyhedrin

Protein TagHis Tag (6x), V5 Epitope Tag

Unit SizeEach

Contents & Storage

The BaculoDirect expression kits include five 0.3 µg vials of BaculoDirect Linear DNA, 125 µL of Cellfectin transfection reagent, frozen Sf9 cells, Gateway LR Clonase enzyme mix, GIBCO Unsupplemented Grace's Insect Medium, and ganciclovir.

Store the BaculoDirect Linear DNA, Cellfectin reagent, and Grace's Medium at +4°C. Store LR Clonase mix at -80°C. Store Sf9 cells in liquid nitrogen. All components are guaranteed stable for 6 months if stored properly.

Frequently asked questions (FAQs)

I cannot grow this white colony in liquid culture. What should I do?

The concentration of gentamicin might be too high. Try lowering the amount to 5 µg/mL and try adding more of the colony to the culture medium.

What has happened when I see blue colonies? How about colonies which are blue in the center and white on the edges?

In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.

I'm getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

I've infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

I'm worried that I am not getting plaques. How many days does it take to see plaques and what size are they typically?

Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.

View all BaculoDirect™ C-Term Expression Kit FAQs

Citations & References (9)

Citations & References

Abstract

Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population.

Authors:Hichiya H, Tanaka-Kagawa T, Soyama A, Jinno H, Koyano S, Katori N, Matsushima E, Uchiyama S, Tokunaga H, Kimura H, Minami N, Katoh M, Sugai K, Goto Y, Tamura T, Yamamoto N, Ohe Y, Kunitoh H, Nokihara H, Yoshida T, Minami H, Saijo N, Ando M, Ozawa S, Saito Y, Sawada J,

Journal:Drug Metab Dispos

PubMed ID:15716363

Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511G>A (G171S), 556C>T (R186X; X represents the translational stop ... More

Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186.

Authors:Li Q, Price JP, Byers SA, Cheng D, Peng J, Price DH,

Journal:J Biol Chem

PubMed ID:15965233

'Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components ... More

HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK.

Authors:Byers SA, Price JP, Cooper JJ, Li Q, Price DH,

Journal:J Biol Chem

PubMed ID:15713662

'The kinase activity of positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1 or T2, is required for the transition of RNA polymerase II into productive elongation. P-TEFb activity has been shown to be negatively regulated by association with the small nuclear RNA 7SK and ... More

Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts.

Authors:Arias EE, Walter JC,

Journal:Genes Dev

PubMed ID:15598982

'In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single ... More

Characterization of Aplysia enticin and temptin, two novel water-borne protein pheromones that act in concert with attractin to stimulate mate attraction.

Authors:Cummins SF, Nichols AE, Amare A, Hummon AB, Sweedler JV, Nagle GT,

Journal:J Biol Chem

PubMed ID:15054104

'Mate attraction in Aplysia involves a long-distance water-borne signal (attractin) that is released during egg laying. Other pheromones are predicted to be released during egg laying that act in concert with albumen gland attractin to stimulate attraction, but their identities are unknown. To identify other candidate water-borne pheromones, we employed ... More

View all BaculoDirect™ C-Term Expression Kit citations