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Related Experiment Videos

Copy-control pBAC/oriV vectors for genomic cloning.

Jadwiga Wild1, Waclaw Szybalski

  • 1McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, USA.

Methods in Molecular Biology (Clifton, N.J.)
|July 23, 2004
PubMed
Summary

This study introduces an improved Bacterial Artificial Chromosome (BAC) system for enhanced DNA cloning and library construction. The new system allows for on-demand amplification of BAC plasmids and cloned genomic DNA, improving efficiency.

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Copy-control tightly regulated expression vectors based on pBAC/oriV.

Methods in molecular biology (Clifton, N.J.)·2004

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Bacterial Artificial Chromosomes (BACs) are essential tools for cloning large DNA fragments.
  • Existing BAC systems have limitations in DNA yield for downstream applications.
  • Efficient amplification of cloned genomic DNA is crucial for library construction and genetic studies.

Purpose of the Study:

  • To describe an improved BAC system for genomic DNA cloning and library construction.
  • To enable on-demand amplification of BAC plasmids and cloned DNA.
  • To enhance the utility of BAC technology for large-scale DNA manipulation.

Main Methods:

  • Development of a modified BAC plasmid (pBAC/oriV) with an additional replication origin (oriV).
  • Utilizing a host strain with an arabinose-inducible promoter (Para) controlling the trfA replicator gene.

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  • Implementing a two-stage replication control system for single-copy maintenance and inducible amplification.

    • Main Results:

    • The improved BAC system retains the advantages of original BACs.
    • BAC plasmids and cloned DNA can be amplified up to 100-fold on command.
    • The system allows for controlled amplification based on insert size and experimental needs.

    Conclusions:

    • The enhanced BAC system provides a significant improvement for genomic DNA cloning and library construction.
    • The inducible amplification feature addresses the need for increased DNA yields.
    • This technology offers a powerful tool for researchers requiring large quantities of specific genomic DNA inserts.