Bridge two double-stranded fragments with a synthetic single-stranded DNA oligo, for simple and fast construction. The bridge is designed as a single-stranded 60-basepair oligo that contains 25 to 30 basepairs of homology with an end of each DNA fragment. The bridge can be customized to include additional sequences, if desired.
During assembly, the T5 exonuclease activity chews back the 5-prime end of the DNA fragment, generating a 3-prime single-stranded overhang, which allows the oligo to anneal with it. The high-fidelity DNA polymerase activity fills the gap, and DNA ligase seals the nicks. The T5 exonuclease activity then cleaves again to allow it to anneal with the overhang on the other DNA fragment. The gaps are once again filled, and nicks are sealed.
Help us celebrate our 50th anniversary! We have hidden 1,000 golden butterflies and are waiting for you to find them. They can be anywhere that you find NEB! Beginning April 15th, be sure to visit our website and tables at tradeshows and events you are attending. Visit our social media channels frequently for tips on where we have hidden the butterflies – and once you find one, either click or scan the code to be eligible for a 50th anniversary prize pack, as well as a grand prize trip to NEB headquarters in Ipswich, MA!.
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