Placeholder Image

Subtitles section Play video

  • 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 CLONEWITHNEB.com 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

Subtitles and vocabulary

Operation of videos Adjust the video here to display the subtitles

B2 cloning dna polymerase pcr gene fragment

Overview of PCR Cloning

  • 61 11
    Cheng-Hong Liu posted on 2014/12/15
Video vocabulary