NEBuilder® HiFi DNA Assembly Cloning Kit

#E5520

Catalog # Size Price (£)
E5520S 10 REACTIONS £173.00

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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. Visit NEBuilderHifi.com for more information.

  • Assembly and transformation in just under two hours

  • Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly

  • Flexible sequence design (scar-less cloning)

  • No PCR clean-up step required

  • Join DNA fragments together more efficiently, even with larger fragments or low DNA inputs

  • Use NEBuilder HiFi in successive rounds of assembly, because it removes 5´ and 3´ end mismatches. (Save time by avoiding time-consuming PCR amplification steps.)

  • Bridge two ds-fragments with a synthetic ss-DNA oligo

    for simple and fast construction (e.g., linker insertion or gRNA library)

  • Use with lower DNA input requirements

  • Flexible sequence design (scar-less cloning)

  • Easily adapted for multiple DNA manipulations, including site-directed mutagenesis

  • Supplied with 10 x 50 μl vials of NEB® 5-alpha Competent E. coli (NEB #C2987) which are chemically competent


NEBuilder HiFi DNA Assembly Cloning Kit 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 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 routine assemblies 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:

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 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

Kit Components

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

  • E5520S

     

     

    Multi-temperature

     

     

Advantages and Features

Properties & Usage

Related Products

Product Notes

FAQs

Quality Control Assays

Certificate of Analysis

Change Notifications

NEBuilder HiFi DNA Assembly Mix has been reformulated to be more stable starting with Lot #10111279 and all subsequent (higher number) lots.

Specifications & Change Notifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

The following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.



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