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  • Traditional cloning evolved from the discoveries of site-specific nucleases (restriction enzymes)

  • and DNA ligases in the 1970s, to enable the era of molecular cloning. At the beginning,

  • cloning a gene into a plasmid allowed the host organism to replicate the cloned sequence

  • and this procedure fuelled the early gene sequencing, protein expression, and gene function

  • studies. The first step is to identify unique restriction

  • sites in your source DNA that can be used to isolate the fragment of interest. We recommend

  • using NEBCutter, an online tool available at NEBCutter.neb.com. In many cases, PCR can

  • be used to add the necessary restriction sites to the gene-of-interest to facilitate directional

  • cloning. Digest your vector and DNA fragment-of-interest

  • with a single restriction enzyme for non-directional cloning, or a pair of restriction enzymes

  • possessing different cleavage sites for directional cloning. Use of two different enzymes that

  • produce blunt ends creates a non-directional cloning strategy. If you are using a single

  • restriction enzyme or two enzymes that produce blunt ends, additional screening may be required.

  • To avoid the possibility of vector self-ligation in the ligation step, it is common practice

  • to dephosphorylate the vector, to remove the 5' phosphate group that the DNA ligase needs

  • for phosphodiester bond formation. Depending on how you generate your vector and insert,

  • other end treatments may be required. These include blunting, A-tailing, and phosphorylation.

  • Select a ligase to covalently join the vector and insert.

  • Transform the recombinant plasmid into competent E. coli.

  • Screen the resulting colonies for the DNA fragment of interest.

  • Although the basic workflow in traditional cloning has not changed much since the 1970s,

  • the introduction of faster and much more robust enzyme and buffer combinations, as well as

  • wider specificities in the last decade has made this approach easier and faster than

  • ever before. Visit CLONEWITHNEB.com for the full list of products available for this application.

Traditional cloning evolved from the discoveries of site-specific nucleases (restriction enzymes)

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B2 中上級

従来のクローニングの概要 (Overview of Traditional Cloning)

  • 28 3
    Cheng-Hong Liu に公開 2021 年 01 月 14 日
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