BaculoDirect™ C-Term Expression Kit

Zitierungen und Referenzen (9)

Invitrogen™

BaculoDirect™ C-Term Expression Kit

Katalognummer	Menge
12562013	5 Transfektionen

Katalognummer 12562013

Preis (EUR)

2.620,00

Zum Warenkorb hinzufügen

Menge:

5 Transfektionen

Preis (EUR)

2.620,00

Zum Warenkorb hinzufügen

Das BaculoDirect™ Baculovirus-Expressionsystem ist ein leistungsstarkes und vielseitiges eukaryotisches System für die hochstufige Proteinexpression in Insektenzellen. Die Kombination von Gateway™-Technologie mit Baculovirus-Expression macht das BaculoDirect-System zur schnellsten und einfachsten Methode für die Produktion von rekombinantem Baculovirus.

Funktionsweise
BaculoDirect™ Lineare DNA (das Baculovirus-Genom) ist so konstruiert, dass sie attR-Stellen für eine schnelle und effiziente Rekombination mit einem Gateway Entry-Klon enthält. Das Gen von Interesse wird mit einer einfachen einstündigen LR-Reaktion vom Entry-Klon in BaculoDirect Linear DNA rekombiniert (Abb. 1). Das resultierende Reaktionsgemisch enthält rekombinantes Baculovirus, das das Gen von Interesse trägt, und wird direkt zur Transfektion von Insektenzellen verwendet Eliminiert die Notwendigkeit, Bakterien zu transformieren und große Bacmid- oder Co-Transfektion von Transfervektor und linearer Baculovirus-DNA in Insektenzellen zu isolieren, um die Bearbeitungszeit zu verkürzen Dadurch verkürzt sich die Einarbeitungszeit erheblich. Gereinigtes Baculovirus kann in weniger als einer Woche isoliert werden.

Gateway lineare DNA
BaculoDirect Lineare DNA ist für schnelles Klonen mit einem Gateway-Entry-Klon und anschließender Expression in Sf9 oder Sf21 Insektenzellen konzipiert. Die lineare DNA umfasst:

• Starker Polyhedrin-Promotor für hohe Expression
• R-Stellen für eine effiziente Rekombination mit jedem attL-flankierten Gateway-Eingangsvektor
• TK-Gen für eine Negativselektion mit Ganciclovir
• C-terminal V5-His-Tag (BaculoDirect™ C-Term Expressionskit) oder N-terminal V5-His-Tag (BaculoDirect™ N-Term Expressionskit) Für den Nachweis mit Anti-V5-Antikörpern und die Aufreinigung mit Nickel-Chelator-Harz
• TEV-Protease-Spaltstelle zur Entfernung des V5-His-Tags nach der Aufreinigung (BaculoDirect N-Term Expressionskit)
• LacZ-Gen, um sicherzustellen, dass ein reiner Baculovirus-Bestand erzeugt wird, der nach der rekombinanten LR-Reaktion durch Ihr interessierendes Gen ersetzt wird

Zusätzliche Materialien erforderlich, separat erhältlich: Gateway-Entry-Klon.

Eine Auswahlhilfe für die Auswahl des am besten geeigneten Gateway-Eingabevektors für Ihre Anwendung finden Sie unter www.thermofisher.com/Gateway.

Nur für Forschungszwecke. Nicht zur Verwendung bei diagnostischen Verfahren.

Specifications

ProdukttypExpressionskit

Menge5 Transfektionen

VektorBaculoDirect-Vektoren

KlonierungsmethodeGateway™

ProduktlinieBaculoDirect, Gateway

PromoterPolyhedrin

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

Unit SizeEach

Inhalt und Lagerung

Die BaculoDirect Expressionskits enthalten fünf 0,3-µg-Fläschchen BaculoDirect Linear DNA, 125 µl Cellfectin Transfektionsreagenz, gefrorene Sf9-Zellen, Gateway LR Clonase Enzymgemisch, GIBCO Unsupplemented Grace's Insect Medium und Ganciclovir.

BaculoDirect Linear DNA, Cellfectin-Reagenz und Grace's Medium bei +4 °C lagern Lagern Sie das LR Clonase-Gemisch bei -80 °C. Lagerung von Sf9-Zellen in Flüssigstickstoff. Alle Komponenten bleiben bei ordnungsgemäßer Lagerung 6 Monate lang haltbar.

Häufig gestellte Fragen (FAQ)

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

Zitierungen und Referenzen (9)

Zitierungen und Referenzen

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

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