Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system
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In plant functional genomic studies, gene cloning into binary vectors for plant transformation is a routine procedure. Traditionally, gene cloning has relied on restriction enzyme digestion and ligation. In recent years, however, Gateway® cloning technology (Invitrogen Co.) has developed a fast and reliable alternative cloning methodology which uses a phage recombination strategy. While many Gateway- compatible vectors are available, we frequently encounter problems in which antibiotic resistance genes for bacterial selection are the same between recombinant vectors. Under these conditions, it is difficult, if not sometimes impossible, to use antibiotic resistance in selecting the desired transformants. We have, therefore, developed a practical procedure to solve this problem.
An integrated protocol for cloning genes of interest from PCR to Agrobacterium transformants via the Gateway® System was developed. The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections.
The protocol presented here is a streamlined procedure for fast and reliable cloning of genes of interest from PCR to Agrobacterium via the Gateway® System. This protocol overcomes a key problem in which two recombinant vectors carry the same antibiotic selection marker. In addition, the protocol could be adapted for high-throughput applications.
© 2008 Xu and Li; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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VCU Anatomy and Neurobiology Publications
Originally published at http://dx.doi.org/10.1186/1746-4811-4-4