Bac-to-Bac™ Baculovirus Expression System
Note: Bac-to-Bac™ Expression System kits now include the NEW! ExpiFectamine Sf Transfection Reagent.
Bac-to-Bac™ Baculovirus Expression System
Gibco™

Bac-to-Bac™ Baculovirus Expression System

The Bac-to-Bac baculovirus expression system enables the efficient production of recombinant baculovirus for expression testing in insect cells. The systemRead more
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Catalog number 10359016
Price (USD)
1,770.00
Each
Add to cart
Price (USD)
1,770.00
Each
Add to cart
The Bac-to-Bac baculovirus expression system enables the efficient production of recombinant baculovirus for expression testing in insect cells. The system relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells. This system features:

Time-saving expression bacmid—With the Bac-to-Bac system, the expression cassette of the pFastBac vector recombines with the parent bacmid in DH10Bac E. coli Competent Cells to form an expression bacmid. The bacmid is then transfected into insect cells for production of recombinant baculovirus particles.

Easy colony screening—The parent bacmid in DH10Bac E. coli contains a segment of the lacZa gene. The lacZa gene is disrupted upon transposition of the expression cassette into the bacmid, allowing for blue/white selection of recombinants for easier identification of recombinant colonies.

High transfection efficiency with ExpiFectamine Sf Transfection Reagent—The Bac-to-Bac TOPO expression kits now come with the next-generation ExpiFectamine Sf Transfection Reagent for efficient DNA transfection in insect cells using fast, flexible protocols. Find out more about this reagent ›

Flexibility—The Bac-to-Bac system comes with a pFastBac vector that contains a large multiple cloning site (MCS) for simplified cloning. Two additional vector formats are offered separately: pFastBac HT vector for production of histidine-tagged recombinant proteins and pFastBac Dual vector for expression of two proteins simultaneously using the p10 and polyhedrin promoters.

High protein expression—The pFastBac vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell line such as Sf9, Sf21, and High Five cells.

For Research Use Only. Not for use in diagnostic procedures.
Specifications
PromoterPolyhedrin
Product TypeBaculovirus Expression System
Protein TagUntagged
Cloning MethodRestriction Enzyme/MCS
Quantity1 kit
VectorpFastBac
Product LineBac-to-Bac™
Unit SizeEach
Contents & Storage
Each Bac-to-Bac Baculovirus Expression System includes:
• pFastBac1 vector (10 μg), store at -20°C
• pFastBac 1-Gus control vector (4 ng), store at -20°C
• MAX Efficiency DH10Bac Chemically Competent E. coli (1 kit with 5 x 0.1 mL), store at -80°C
• ExpiFectamine Sf Transfection Reagent (1 mL), store at 4°C, do not freeze

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.