Abstract
T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.
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Abbreviations
- Ampr :
-
Ampicillin resistance gene
- Kmr :
-
Kanamycin resistance gene
- pBR322 origin:
-
pBR322-derived replication origin for E. coli
- pUC origin:
-
pUC-derived replication origin for E. coli
- 2 μ origin:
-
2 μ yeast replication origin
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Acknowledgments
This work was supported in part by a grant-in-aid for scientific research (C) from the Japan Society for the Promotion of Science to Y. N. (17580082) and T. A. (17580008). E. I. is the recipient of research fellowships from the Japan Society for the Promotion of Science for Young Scientists.
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Communicated by H. Ebinuma.
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Nagano, Y., Takao, S., Kudo, T. et al. Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation. Plant Cell Rep 26, 2111–2117 (2007). https://doi.org/10.1007/s00299-007-0428-2
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DOI: https://doi.org/10.1007/s00299-007-0428-2