A One Pot, One Step, Precision Cloning Method with High Throughput Capability
Author Information
Author(s): Carola Engler, Romy Kandzia, Sylvestre Marillonnet, Hany A. El-Shemy
Primary Institution: Icon Genetics GmbH, Biozentrum Halle, Halle, Germany
Hypothesis
Can a new subcloning strategy using type IIs restriction enzymes improve the efficiency and precision of DNA cloning?
Conclusion
The new 'Golden Gate' cloning method allows for nearly 100% correct recombinant plasmids to be obtained in a single step without unwanted sequences.
Supporting Evidence
- The method allows for the cloning of multiple DNA fragments in a single tube.
- Cloning efficiency increases with incubation time, but 5 minutes is sufficient for most cases.
- All tested constructs showed the expected sequences at the junction sites after cloning.
Takeaway
This study shows a new way to clone DNA that is faster and doesn't leave extra bits of DNA in the final product, making it cleaner and more precise.
Methodology
The method uses type IIs restriction enzymes for a one-step restriction-ligation process to create recombinant plasmids.
Limitations
The presence of internal BsaI restriction sites in the gene of interest can complicate the cloning process.
Digital Object Identifier (DOI)
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