Format

Send to:

Choose Destination
See comment in PubMed Commons below
PLoS One. 2012;7(5):e37429. doi: 10.1371/journal.pone.0037429. Epub 2012 May 18.

Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

Author information

  • 1School of Biotechnology, KTH-Royal Institute of Technology, Stockholm, Sweden.

Abstract

In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

PMID:
22624028
[PubMed - indexed for MEDLINE]
PMCID:
PMC3356258
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Public Library of Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk