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Related Experiment Videos

The pCLIP plasmids: versatile cloning vectors based on the bacteriophage lambda origin of replication

A C Boyd1, D J Sherratt

  • 1MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK.

Gene
|February 3, 1995
PubMed
Summary

New pCLIP plasmids offer versatile cloning and reversible integration into lambda phage, enabling manipulation of DNA in plasmid or phage forms. These tools enhance genetic engineering flexibility for researchers.

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Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Recombinant DNA Technology

Background:

  • Development of versatile cloning vectors is crucial for molecular biology research.
  • Existing vectors may lack flexibility in DNA maintenance and manipulation.
  • Phage lambda origin of replication offers unique integration capabilities.

Purpose of the Study:

  • To construct and characterize a novel series of general-purpose plasmid vectors.
  • To enhance cloning potential and introduce reversible integration into phage lambda.
  • To provide researchers with flexible tools for DNA manipulation in various forms.

Main Methods:

  • Construction of plasmid vectors (pCLIP series) using phage lambda origin of replication.
  • Insertion of lacZ alpha-complementing multiple cloning site (MCS) and removal of superfluous restriction sites.

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  • Demonstration of reversible integration into lambda prophage via homologous recombination.
  • Production and manipulation of phage::plasmid hybrids.

    • Main Results:

    • Successfully constructed pCLIP plasmids (pCLIP8, pCLIP9, pCLIP18, pCLIP19, pCLIP23) with medium copy number and broad compatibility.
    • pCLIP vectors offer 17 unique restriction sites for enhanced cloning potential.
    • Demonstrated reversible integration into lambda prophage, allowing DNA maintenance in single or multiple copy.
    • Successfully generated and manipulated phage::plasmid hybrids using a test recombinant plasmid (pCLIPLEU2).

    Conclusions:

    • The pCLIP plasmid vectors provide a robust platform for routine cloning and complementation analysis.
    • Their unique ability to integrate reversibly into lambda prophage offers unprecedented flexibility in DNA manipulation.
    • These vectors are advantageous for applications requiring manipulation of recombinants in plasmid, phage, or prophage forms.