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- Title
Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
- Authors
Hudson, Elton P; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan
- 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.
- Publication
PloS one, 2012, Vol 7, Issue 5, pe37429
- ISSN
1932-6203
- Publication type
Journal Article
- DOI
10.1371/journal.pone.0037429