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.
Original language | English |
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Journal | P L o S One |
Volume | 7 |
Issue number | 5 |
Pages (from-to) | - |
ISSN | 1932-6203 |
DOIs | |
Publication status | Published - 2012 |
Keywords
- BIOLOGY
- DNA-MOLECULES
- PCR PRODUCTS
- PROTEIN EXPRESSION
- PICHIA-PASTORIS
- IN-VITRO
- CLONING
- RECOMBINATION
- HYBRIDIZATION
- EMULSION
- TARGETS