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  • PCR cloning is a common approach for the cloning of any DNA fragment of interest. This approach

  • does not require of a lot of input DNA and allows for the cloning of fragments for which

  • the whole sequence may not be known. Begin by amplifying the DNA fragment of interest

  • with a polymerase using primers that are complementary to the DNA sequence of interest. With each

  • cycle, the resulting DNA is amplified exponentially. The resulting amplified DNA will have either

  • a single-A overhang, if Taq DNA polymerase was used, or a blunt end, if a high-fidelity

  • polymerase, such as Q5 High-Fidelity DNA Polymerase, was used.

  • Next, join the amplified DNA fragments to the appropriate vector. This can be done by

  • the use of a DNA ligase, or by use of activated vectors that are covalently-bound to an effector

  • enzyme, which facilitates vector:insert joining. Depending on the vector chosen, the fragment

  • of interest may be inserted non-directionally at the ligation junction, such as in TA cloning.

  • Some supplied vectors can contain a toxic gene. The use of toxic gene fusions ensures

  • that the only competent cells that survive are those that have taken up the vectors with

  • successful insertion of the target gene sequence, as the insertion disrupts the expression of

  • the toxic gene. These vectors are known assuicide vectors”.

  • Transform the recombinant plasmid into competent E. coli. Spread onto agar plates that contain

  • the appropriate antibiotic for selection. Screen the resulting colonies for the gene

  • or fragment of interest. In recent years, the introduction of fast

  • and robust high-fidelity polymerases has made this approach easy and reliable.

  • The NEB PCR Cloning Kit allows quick and simple cloning of all your PCR amplicons, regardless

  • of the polymerase used. This kit utilizes a novel mechanism for background colony suppression,

  • and allows for direct cloning from your reaction, with no purification step.

  • Visit for a full list of products available for this application.

PCR cloning is a common approach for the cloning of any DNA fragment of interest. This approach


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B2 中上級

PCRクローニングの概要 (Overview of PCR Cloning)

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    Cheng-Hong Liu に公開 2021 年 01 月 14 日