字幕列表 影片播放 列印英文字幕 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 as “suicide 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.