The pBC vectors were derived from the pBluescript II phagemid. The ampicillin-resistance gene has been replaced with the chloramphenicol-resistance gene. pBC phagemids (plasmids with a phage origin) are cloning vectors designed to simplify commonly used cloning and sequencing procedures, including the construction of nested deletions for DNA sequencing, generation of RNA transcripts in vitro and site-specific mutagenesis and gene mapping. The pBC phagemids have an extensive polylinker with 21 unique restriction enzyme recognition sites. Flanking the polylinkers are T7 and T3 RNA polymerase promotors that can be used to synthesize RNA in vitro. The choice of promotor used to initiate transcription determines which strand of the insert cloned into the polylinker will be transcribed.
The polylinker and T7 and T3 RNA polymerase promotor sequences are present in the N-terminal portion of a lacZ gene fragment. There are 36 amino acids from the MET sequence to the EcoR I site in the pBC plasmid. There are a total of 131 amino acids, but this is interrupted by the large polylinker. pBC phagemids having no inserts in the polylinker will grow as blue colonies on the appropriate strains of bacteria (i.e., strains containing the lacZ?M15 on an F´ episome, XL1-Blue MRF´, among others). pBC phagemids which have inserts will grow as white colonies on the same strain, because the inserts disrupt the coding region of the lacZ gene fragment.
pBC (+) and (–) are available with two polylinker orientations designated as either KS or SK using the following convention: (1) in the KS orientation, the Kpn I restriction site is nearest the lacZ promotor and the Sac I restriction site is farthest from the lacZ promotor; and (2) in the SK orientation, the Sac I site is the closest restriction site to the lacZ promotor and the Kpn I site is the farthest.
Flanking the T3 and T7 promotors are BssH II sites. This rare six-base cutter will allow the insert plus the T phage RNA promotors to be excised and used for gene mapping.
pBC phagemids can be rescued as single-stranded (ss) DNA. pBC phagemids contain a 454-nucleotide filamentous f1 phage intergenic region (M13 related). The (+) and (–) orientations of the f1 intergenic region allow the rescue of sense or antisense ssDNA by a helper phage. This ssDNA can be used for dideoxynucleotide sequencing (Sanger method) or site-specific mutagenesis.
Note: We have discovered that the use of excess amounts of EcoR I to digest pBC results in EcoR I prime activity. This appears to cut at a non-EcoR I site at the 3´ end of the f1 intergenic region causing confusion when interpreting results from an agarose gel. If a restriction pattern appears incorrect, try reducing the units of EcoR I and check whether a normal restriction pattern is restored.
For Research Use Only. Not for use in diagnostic procedures