Bac-to-Bac™ Vector Kit
New—ExpiSf™ Expression System for superior protein yields from insect cells. Learn more »
Get your genes cloned into this vector with GeneArt Gene Synthesis Services
Bac-to-Bac™ Vector Kit
Gibco™

Bac-to-Bac™ Vector Kit

The Bac-to-Bac™ Vector Kit contains a pFastBac™ 1 vector, as well an expression control vector, intended for use as partRead more
Have Questions?
Catalog number 10360014
Price (USD)
1,132.00
Each
Add to cart
Price (USD)
1,132.00
Each
Add to cart
The Bac-to-Bac™ Vector Kit contains a pFastBac™ 1 vector, as well an expression control vector, intended for use as part of the Bac-to-Bac™ Baculovirus Expression System (Cat. No. 10359-016), which enables the efficent production of recombinant baculovirus for expression testing in insect cells. The Bac-to-Bac™ System relies on generation of recombinant baculovirus by site-specific transposition in E. colirather than homologous recombination in insect cells (Figure 1). This system features:

• Time-saving expression bacmid. With Bac-to-Bac™, the expression cassette of the pFastBac™ vector recombines with the parent bacmid in DH10Bac™ E. coli Competent Cells (not included with this vector kit) 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. colicontains a segment of the lacZα gene. The lacZα gene is disrupted upon transposition of the expression cassette into the bacmid allowing for blue/white selection of recombinants. This makes identification of recombinant colonies easy.
The Bac-to-Bac™ Baculovirus Expression System is designed for fast, small-scale production of recombinant baculovirus. The pFastBac™ 1 vector provided by this kit offers the strong polyhedrin promoter for protein expression and a large multiple cloning site for simplified cloning.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
PromoterPolyhedrin
Product TypeVector Kit
Protein TagUntagged
Cloning MethodRestriction Enzyme/MCS
Quantity1 kit
VectorpFastBac
Product LineBac-to-Bac™
Unit SizeEach
Contents & Storage
Contains 10 μg of pFastBac™1 vector and pFastBac™ 1-Gus control vector.

Store at -20°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.

View all Bac-to-Bac™ Vector Kit FAQs