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Related Experiment Videos

DNA sequencing from single phage plaques using solid-phase magnetic capture

S Wang1, M Krinks, M Moos

  • 1Food and Drug Administration, Rockville, MD.

Biotechniques
|January 1, 1995
PubMed
Summary
This summary is machine-generated.

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This study introduces a rapid method for sequencing lambda phage DNA, saving time in molecular cloning. The new strategy provides clear DNA sequence data without subcloning, improving gene family identification.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Molecular cloning operations, particularly subcloning cDNA from lambda gt11 phage into plasmid vectors, are often labor-intensive and time-consuming.
  • Assessing insert size and sequence unambiguously without subcloning is crucial for efficiency, especially when identifying multiple gene family members using degenerate PCR or low-stringency hybridization.

Purpose of the Study:

  • To develop a simple, reliable, and efficient strategy for sequencing small amounts of lambda phage DNA, lysates, or individual phage plaques.
  • To provide an alternative to traditional thermal cycle sequencing with improved clarity and speed.

Main Methods:

  • Utilizing universal lambda phage primers and rapid air thermal cycling.
  • Employing streptavidin magnetic bead capture for highly purified single-stranded DNA templates.

Related Experiment Videos

  • Leveraging T7 DNA polymerase for high-clarity DNA sequencing.

    • Main Results:

    • Routine generation of 350-500 bases of unambiguous sequence per reaction.
    • Completion of sequencing reactions within hours of lifting phage plaques.
    • Obtained sequence data with significantly higher clarity compared to standard thermal cycle sequencing.

    Conclusions:

    • The described strategy offers an efficient and rapid method for DNA sequencing of lambda phage clones.
    • This approach significantly reduces the time and labor associated with molecular cloning and gene family analysis.
    • The method provides a valuable alternative for obtaining high-quality sequence data from lambda phage constructs.