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Related Experiment Videos

Subcloning using simplified adaptor addition.

J M Supak-Koslovsky1, M D Thomas

  • 1Department of Plant Pathology and Microbiology, Texas A&M University College Station 77843.

Biotechniques
|August 1, 1992
PubMed
Summary
This summary is machine-generated.

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This study presents a simplified DNA cloning method using synthetic oligonucleotide adaptors. The new procedure streamlines the process by eliminating extra enzymatic steps, making DNA fragment subcloning more efficient and reliable.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Enzymology

Background:

  • Cloning DNA fragments with incompatible ends traditionally involves complex multi-step procedures.
  • Excess synthetic oligonucleotide adaptors often require removal via enzymatic digestion or extensive purification.
  • The organophosphate degradation (opd) gene from Flavobacterium sp. encodes parathion hydrolase.

Purpose of the Study:

  • To develop a simplified method for adding synthetic oligonucleotide adaptors to DNA fragments for subcloning.
  • To eliminate the need for enzymatic digestion to remove excess adaptors.
  • To improve the efficiency and reliability of DNA fragment cloning.

Main Methods:

  • A simplified procedure for adding synthetic oligonucleotide adaptors to DNA fragments with incompatible ends.

Related Experiment Videos

  • Cloning of the organophosphate degradation (opd) gene into the fungal vector pH1S via the HindIII site.
  • All enzymatic steps performed in a single microcentrifuge tube to minimize DNA loss.

    • Main Results:

    • The opd gene was successfully cloned into the pH1S vector, with 12% of recovered plasmids containing the insert.
    • The opd gene was inserted in both orientations with similar frequencies (53% and 47%).
    • The simplified procedure avoided traditional purification steps like phenol/chloroform extraction and ethanol precipitation.

    Conclusions:

    • The presented method offers a simpler and more reliable approach to DNA fragment subcloning using synthetic oligonucleotide adaptors.
    • Eliminating intermediate purification steps enhances procedural efficiency and reduces potential DNA loss.
    • This technique facilitates the cloning of specific genes, such as the opd gene, into expression vectors.