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Reproductive cloning is the process of producing a genetically identical copy—a clone—of an entire organism. While clones can be produced by splitting an early embryo—similar to what happens naturally with identical twins—cloning of adult animals is usually done by a process called somatic cell nuclear transfer (SCNT).
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The first successfully cloned mammal was Dolly, a sheep, born on 5th July 1996 at Roslin Institute, Scotland. The cloned sheep was named after the American singer Dolly Parton. Dolly lived for seven years and died of respiratory complications, which is speculated to be due to the actual age of her DNA. Because the DNA in cloned cells belongs to an older individual,  the cloned individual’s life expectancy may be affected. Indeed, analysis of Dolly’s DNA revealed shorter...
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Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
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Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

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Golden Gate cloning.

Carola Engler1, Sylvestre Marillonnet

  • 1Icon Genetic GmbH, Halle, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|January 8, 2014
PubMed
Summary
This summary is machine-generated.

Golden Gate cloning enables rapid DNA assembly of up to nine fragments in one step. This protocol details plasmid design and reaction conditions for efficient molecular cloning and biotechnology applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Synthetic Biology

Background:

  • DNA assembly is crucial for biological research and biotechnology.
  • Conventional cloning methods are often multi-step and time-consuming.
  • Newer methods aim to streamline and accelerate DNA fragment assembly.

Purpose of the Study:

  • To provide a protocol for designing plasmids for Golden Gate cloning.
  • To describe the necessary conditions for performing Golden Gate assembly reactions.
  • To facilitate efficient and rapid DNA construct generation.

Main Methods:

  • Golden Gate cloning utilizes a type IIS restriction enzyme and ligase.
  • Assembly involves combining multiple DNA fragments and a recipient vector in a single tube.
  • Incubation in a thermal cycler facilitates the reaction.

Main Results:

  • Golden Gate cloning allows assembling up to nine DNA fragments simultaneously.
  • The majority of resulting clones contain the desired construct after transformation.
  • Careful design of donor and recipient plasmids is essential for success.

Conclusions:

  • Golden Gate cloning offers a simplified and efficient method for DNA assembly.
  • This protocol enables researchers to design necessary plasmids and optimize reaction conditions.
  • The method significantly speeds up the generation of complex DNA constructs.