(USA Customers: Please note you will receive two separate boxes when you order this product; one box will contain the competent cells on dry ice; the other box will contain NEBuilder HiFi DNA Assembly Master Mix on wet ice.)NEB recommends NEB 10-beta Competent E. coli (HighEfficiency, NEB #C3019)for assemblies larger than 15 kb. NEBuilder HiFi DNA Assembly Bundle for Large Fragments offers a discountfor purchasing these competent cells with NEBuilder HiFi DNA AssemblyMaster Mix.NEBuilder HiFi DNA Assembly 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 worktogether 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 Cloning Kit is supplied with NEB 5-alpha High Efficiency Competent 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 routineassemblies of 15kb 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 15kb. 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

This product is related to the following categories:

DNA Assembly, Cloning and Mutagenesis Kits Products

This product can be used in the following applications:

NEBuilder® HiFi DNA Assembly,

Genome Editing Applications

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