Invitrogen Bac to Bac Expression System

Generating the Recombinant pFastBac Vector
General Information
Introduction
To generate a recombinant plasmid containing your gene(s) of interest for use in
the Bac-to-Bac ® Baculovirus Expression System, you will use restriction enzyme
digestion and ligation to clone your gene(s) into one of the pFastBac vectors. For
recommendations and guidelines to help you design your cloning strategy, refer to
the appropriate section on pages 10-16 depending on the pFastBac vector you are
using.
General Molecular
Biology
Techniques
For help with restriction enzyme digestion, ligation, DNA sequencing, and other
general molecular biology techniques, refer to Molecular Cloning: A Laboratory
Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et
al., 1994).
Propagation and
Maintenance of
Plasmids
The pFastBac vectors and their corresponding expression control plasmids
contain the ampicillin resistance gene to allow for selection in E. coli using
ampicillin. To propagate and maintain the pFastBac vectors and the pFastBac
control plasmids, use the following procedure:
1. Use the stock solution of vector provided to transform a recA, endA E. coli
strain such as TOP10, DH10B , or DH5α (see page 17 for more information).
2. Select transformants on LB agar plates containing 100 µg/ml ampicillin.
3. Prepare a glycerol stock from each transformant containing plasmid for longterm
storage (see page 18).
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