Bac-to-Bac™ Vector Kit

Zitierungen und Referenzen (12)

Gibco™

Bac-to-Bac™ Vector Kit

Katalognummer	Menge
10360014	1 Kit

Katalognummer 10360014

Preis (EUR)

1.026,00

Zum Warenkorb hinzufügen

Menge:

1 Kit

Preis (EUR)

1.026,00

Zum Warenkorb hinzufügen

Das Bac-to-Bac™ Vektor-Kit enthält einen pFastBac™1 Vektor sowie einen Expressionskontrollvektor, der als Teil des Bac-to-Bac™ Baculovirus-Expressionssystems (Kat. Nr. 10359-016) die effiziente Produktion von rekombinantem Baculovirus für Expressionstests in Insektenzellen ermöglicht. Das Bac-zu-Bac™ -System beruht eher auf der Erzeugung rekombinanter Baculoviren durch stellenspezifische Transposition in E. coli als auf homologer Rekombination in Insektenzellen (Abb. 1). Dieses System hat folgende Merkmale:

• Zeitsparender Expressions-Bacmid. Mit Bac-to-Bac™ rekombiniert die Expressionskassette des pFastBac™ Vektors mit dem Parental-Bacmid in DH10Bac™ E. coli Kompetenten Zellen (nicht im Lieferumfang dieses Vektor-Kits enthalten), um ein Expressions-Bacmid zu bilden. Das Bacmid wird dann in Insektenzellen zur Produktion rekombinanter Baculovirus-Partikel transfiziert.
• Einfaches Kolonie-Screening. Das Parental-Bacmid in DH10Bac™ E. coli enthält ein Segment des lacZα-Gens. Das lacZα-Gen wird bei der Umsetzung der Expressionskassette in das Bacmid gestört, wodurch eine blau/weiße Selektion von Rekombinanten ermöglicht wird. Dies erleichtert die Identifizierung von rekombinanten Kolonien.
Das Bac-to-Bac™ Baculovirus-Expressionssystem wurde für die schnelle, kleine Produktion von rekombinantem Baculovirus entwickelt. Der von diesem Kit bereitgestellte pFastBac™ 1 Vektor verfügt über einen starken Polyedrin-Promotor für die Proteinexpression und eine große Stelle für mehrere Klonierungen zum einfacheren Klonieren.

Nur für Forschungszwecke. Darf nicht für diagnostische Verfahren eingesetzt werden.

Specifications

ProdukttypVektorkit

Menge1 Kit

VektorpFastBac

KlonierungsmethodeRestriktionsenzym/MCS

ProduktlinieBac-to-Bac

PromoterPolyhedrin

ProteinmarkierungNicht markiert

Unit SizeEach

Inhalt und Lagerung

Enthält 10 µg pFastBac™1-Vektor und pFastBac™ 1-Gus Kontrollvektor.

Lagerung bei -20 °C.

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 Bac-to-Bac™ Vector Kit FAQs

Zitierungen und Referenzen (12)

Zitierungen und Referenzen

Abstract

Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin.

Authors: Dan C; Kelly A; Bernard O; Minden A;

Journal:J Biol Chem

PubMed ID:11413130

'PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in ... More

Allocation of helper T-cell epitope immunodominance according to three-dimensional structure in the human immunodeficiency virus type I envelope glycoprotein gp120.

Authors: Dai G; Steede N K; Landry S J;

Journal:J Biol Chem

PubMed ID:11551929

'The specificity and intensity of CD4(+) helper T-cell responses determine the effectiveness of immune effector functions. Promiscuously immunodominant helper T-cell epitopes in the human immunodeficiency virus (HIV) envelope glycoprotein gp120 could be important in the development of broadly protective immunity, but the underlying mechanisms of immunodominance and promiscuity remain poorly ... More

Interaction of Fibroblast Growth Factor Receptor 3 and the Adapter Protein SH2-B. A ROLE IN STAT5 ACTIVATION.

Authors: Kong Monica; Wang Ching S; Donoghue Daniel J;

Journal:J Biol Chem

PubMed ID:11827956

'Fibroblast growth factor receptor 3 (FGFR3) influences a diverse array of biological processes, including cell growth, differentiation, and migration. Activating mutations in FGFR3 are associated with multiple myeloma, cervical carcinoma, and bladder cancer. To identify proteins that interact with FGFR3 and which may mediate FGFR3-dependent signaling, a yeast two-hybrid screen ... More

Functional differences between the human ATP-dependent nucleosome remodeling proteins BRG1 and SNF2H.

Authors: Aalfs J D; Narlikar G J; Kingston R E;

Journal:J Biol Chem

PubMed ID:11435432

'ATP-dependent nucleosome remodeling complexes can be grouped into several classes that may differ in their biochemical remodeling activities and biological roles. Although there are a number of biochemical studies of each class of remodeler, there are very little data directly comparing the biochemical activities of remodelers from different classes. We ... More

PIAS1 and PIASxalpha function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription.

Authors:Nishida T, Yasuda H,

Journal:J Biol Chem

PubMed ID:12177000

The androgen receptor (AR) has been shown to be modified by SUMO-1, a ubiquitin-like protein. Recently we showed that PIAS family proteins function as SUMO-E3 ligases. Here we provide evidence that PIAS1 and PIASxalpha act as specific SUMO-E3 ligases for the AR. PIAS1 and PIASxalpha but not PIAS3 or PIASxbeta ... More

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