Bac-to-Bac™ HBM TOPO™ Cloning Kit
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Bac-to-Bac™ HBM TOPO™ Cloning Kit
Gibco™

Bac-to-Bac™ HBM TOPO™ Cloning Kit

Bac-to-Bac™ Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies onRead more
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Catalog number A11338
Price (USD)
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Bac-to-Bac™ Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli (DH10Bac™) rather than homologous recombination in insect cells. The new pFastBac™ HBM TOPO™ vector enables secreted protein expression because it has the honeybee melittin (HBM) secretion signal. The new vector should be strongly considered for the expression of glycoproteins. Glycoproteins cannot be glycosylated in the absence of any secretion signal. In contrast to glycoproteins secreted from mammalian cells, glycoproteins secreted from baculovirus can be easily de-glycosylated in vitro. This is a very important feature in order to crystallize proteins!

The pFastBac™ HBM TOPO™ vector has also a C-terminal His-Tag with a TEV cleavage signal to enable easy purification of Histidine fusion proteins on nickel-chelating resins (ProBond™ Purification System) and native proteins with the aid of AcTev™ protease.
The vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Mimic™ Sf9, Sf21, and High Five™ cells in Sf-900 II & Sf-900™ III media from Gibco.

Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac™'s pFastBac™ vectors, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Cell LineHigh Five™, Sf21, Sf9
Cell TypeInsect Cell
Cloning MethodBlunt TOPO
CleavageTEV Protease Recognition Site
FormatKit
Expression MechanismCell-Based Expression
Expression SystemBaculovirus
For Use With (Application)Protein Expression
High-throughput CompatibilityHigh-throughput Compatible, Not High-throughput Compatible (Manual)
Key FunctionsSecreted Expression
No. of Reactions20 Reactions
Product LineBac-to-Bac™, TOPO™
Product TypeCloning Kit
PromoterPolyhedrin
Protein TagHBM (Honeybee Melittin), His Tag (6x)
Quantity20 Reactions
Selection Agent (Eukaryotic)None
VectorpFastBac/HBM-TOPO
Unit SizeEach
Contents & Storage
Bac-to-Bac™ HBM TOPO™ Cloning Kit contains: (For details see the manual)
• Vector kit for 20 topo cloning reactions
• pFastBac⁄HBM TOPO™ Vector containing the C-terminal TEV cleavage sit and His-Tag.
• pFastBac⁄CT-Gus Control Plasmid (Control expression vector)
• Other reagents supplied: 10x PCR, dNTP Mix, Salt Solution, Sterile Water, Control PCR template, Polyhedrin Forward Primer, SV40 pA Reverse Primer
• Kit containing competent cells (20 reactions):
• One Shot™ Mach1-T1R Chemically Competent E. coli

Vector kit: store at -20°C
One Shot™ Mach1-T1R Chemically Competent E. coli: store at -80°C

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.