NEBuilder HiFi DNA Assembly Master Mix was developed to improve the efficiency and accuracy of DNA assembly. This method allows for seamless assembly of multiple DNA fragments, regardless of fragment length or end compatibility. This method has been used to assemble either single-stranded oligonucleotides or different sizes of DNA fragments with varied overlaps (15–30 bp). It has utility for the synthetic biology community, as well as those interested in one-step cloning of multiple fragments due to its ease of use, flexibility and simple master-mix format. The reaction includes different enzymes that work together in the same buffer (see Figure 1):

• The exonuclease creates single-stranded 3′ overhangs that facilitate the annealing of fragments that share complementarity at one end (the overlap region)
• The polymerase fills in gaps within each annealed fragment
• The DNA ligase seals nicks in the assembled DNA 

The end result is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation of E coli.

NEBuilder HiFi DNA Assembly kits are available in various formats: with NEB 5-alpha chemically competent cells (Cloning Kit, NEB #E5520 ), as a bundle with NEB 10-beta chemically competent cells (Bundle for Large Fragments, NEB #E2623 ) and without competent cells (Master Mix, NEB #E2621 ) . NEB 5-alpha chemically competent cells are excellent for routine assemblies of 15 kb or less. NEB recommends NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019  ) or NEB 10-beta Electrocompetent E. coli (NEB #C3020 ) for assemblies larger than 15 kb. If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040 ) should be used. 

NEBuilder has been used in various applications, including:

• Site-directed mutagenesis
• Construction of an sgRNA-Cas9 expression vector  | Animation
• Assembly of linear yeast expression cassettes

To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart .

For help designing your primers for use with NEBuilder, please view our primer design video .

Figure 1: Not your average DNA assembly reagent



Figure 2: Overview of the NEBuilder HiFi DNA Assembly method



Figure 3: NEBuilder HiFi DNA Assembly offers improved efficiency in 4-fragment assembly reactions


Four fragments (~20 fmol) with 20 bp overlap were assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621 ), GeneArt^® Gibson Assembly^® Mix (Thermo Fisher #A46627) and In-Fusion^® Snap Assembly Master Mix (Takara Bio USA #638947) to create a pUC19 vector. Assembly reactions were performed at 50°C for 60 min or 15 min. 2 μl of each assembled mix was transformed into NEB 5-alpha Competent E.coli (NEB #C2987 ) and spread on LB/Amp plates with IPTG and X-Gal. Blue colonies ( >95% of total colonies) that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Mix yields more colonies than both competitors.

View additional performance data compared to GeneArt Gibson Assembly and In-Fusion Snap Assembly 产品类别: DNA Assembly, Cloning and Mutagenesis Kits Products 应用: NEBuilder® HiFi DNA Assembly,  High-throughput cloning and automation solutions,  Genome Editing Applications