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Related Experiment Videos

Simultaneous cloning of open reading frames into several different expression vectors.

Clement A Stanyon1, Thawornchai Limjindaporn, Russell L Finley

  • 1Wayne State University, Detroit, MI, USA.

Biotechniques
|September 30, 2003
PubMed
Summary

This study introduces a novel in vitro recombination system for simultaneously transferring a single open reading frame (ORF) to four different expression vectors. This method streamlines high-throughput cloning for proteomic studies.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Genomic sequencing has identified numerous genes with unknown functions.
  • High-throughput methods are crucial for determining protein function.
  • Current methods often require multiple cloning steps into various expression vectors.

Purpose of the Study:

  • To develop a method for simultaneously transferring a single open reading frame (ORF) into multiple expression vectors.
  • To facilitate high-throughput subcloning and proteomic research.
  • To create new expression vectors for yeast, including those for the LexA two-hybrid system.

Main Methods:

  • Utilized a commercially available in vitro recombination system.
  • Simultaneously transferred a single ORF from a source vector to four distinct target vectors.

Related Experiment Videos

  • Constructed new yeast expression vectors, including LexA two-hybrid vectors.

    • Main Results:

    • Demonstrated efficient transfer of individual ORFs to four different vectors in a single in vitro reaction.
    • Successfully separated resulting expression plasmids using vector-specific prototrophic markers.
    • Validated the system for producing multiple expression constructs concurrently.

    Conclusions:

    • The developed in vitro recombination system enables simultaneous transfer of ORFs to multiple vectors.
    • This approach significantly enhances efficiency in high-throughput subcloning.
    • The method holds great potential for advancing proteomic studies and functional genomics.