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Related Experiment Videos

Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method.

V Asundi1, B Tyler, K Dreher

  • 1Weis Center for Research, Geisinger Clinic, Danville, PA 17822.

Biotechniques
|November 1, 1990
PubMed
Summary

This study introduces a rapid method for isolating bacteriophage lambda DNA and identifying large cDNA inserts. The technique efficiently screens lambda gt11 cDNA libraries, reducing false positives and saving time in molecular cloning.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Screening cDNA libraries is crucial for gene discovery.
  • Traditional methods for identifying recombinant clones can be laborious and time-consuming.
  • Bacteriophage lambda vectors are widely used for constructing genomic and cDNA libraries.

Purpose of the Study:

  • To develop a rapid and efficient method for isolating bacteriophage lambda DNA.
  • To enable quick identification of large cDNA inserts within crude phage lysates.
  • To streamline the screening process of lambda gt11 cDNA libraries.

Main Methods:

  • Utilized a 32P-radiolabeled cDNA probe for initial screening of a lambda gt11 cDNA library.
  • Employed phage "spot-blot" analysis to rapidly screen putative positive clones.

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  • Applied a modified plate lysate procedure for DNA isolation from crude lysates.
  • Performed EcoRI digestion and Southern blot analysis for examining cDNA inserts.

    • Main Results:

    • Identified 21 putative positive clones from the initial library screening.
    • Phage spot-blot analysis successfully eliminated 9 false positive signals.
    • Isolated and analyzed DNA from 12 recombinant phage clones.
    • Identified two clones with the longest cDNA inserts from a mixed phage population.

    Conclusions:

    • The developed method is rapid, sensitive, reproducible, and cost-effective.
    • It allows simultaneous processing of multiple clones without compromising DNA quality or yield.
    • The procedure obviates the need for extensive plaque purification of all initial positive clones.