Find out how NEBuilder® HiFi DNA Assembly can reliably join DNA fragments in a single tube, isothermal reaction, with advantages over NEB Gibson Assembly®.


NEBuilder® HiFi DNA Assembly, developed by New England Biolabs, is an effective method for the high-fidelity assembly of multiple DNA fragments.

Advantages over Gibson Assembly Master Mix include higher efficiency, higher accuracy, the ability to assemble both 5´- and 3´-restriction enzyme mismatches, the ability to bridge two double-stranded DNA fragments with single-stranded DNA and no licensing fees from NEB.

This is accomplished in a single-tube isothermal reaction with NEBuilder HiFi DNA Assembly Master Mix. The method utilizes adjacent DNA fragments with complementary ends, which can be added, for example, by PCR. Then, the overlapping fragments are added to the NEBuilder HiFi DNA Assembly Master Mix and incubated for 15 minutes to one hour at 50°C.

During the one-hour incubation, the Master Mix’s three enzyme activities set to work on the fragments. First, a 5´ to 3´ exonuclease activity creates single stranded 3´ overhangs. These complementary sequences then anneal, creating the double-stranded DNA of interest. A high-fidelity DNA polymerase then extends the 3´ ends, filling in the gaps, and a DNA ligase seals the remaining nicks. The end result is a fully-sealed, double-stranded DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation.

In addition to joining two double-stranded DNA fragments together with an overlap, NEBuilder HiFi DNA Assembly Master Mix can also join two non-overlapping double-stranded DNA together with an overlapping single-stranded oligonucleotide. NEBuilder HiFi DNA Assembly has been successfully used to reliably join up to six DNA fragments into a single molecule, as well as create a library (gRNA or linker insertion).

This remarkably straightforward and powerful technique makes quick work of large multi-fragment assemblies, but it is also useful for more routine applications, such as cloning. The NEBuilder HiFi DNA Assembly Cloning Kit, which includes both NEBuilder HiFi DNA Assembly Master Mix and NEB 5-alpha competent cells, has been optimized for efficient assembly and cloning of multiple fragments into any vector in just over an hour.

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